Sweet protein from truffle

ABSTRACT

Newly identified fungal sweet-taste modifying proteins, and the cDNA encoding said proteins are described. Specifically, Myd proteins active in sweet taste activation, and the cDNA encoding the same, are described, along with methods for isolating such cDNA and for isolating and expressing such proteins. Also disclosed is use of a sweetening composition which includes the proteins of the invention, and methods to provide improved flavor to a product for oral administration.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application claims the benefit of U.S. Provisional PatentApplication No. 63/044,245, filed Jun. 25, 2020, which is incorporatedby reference.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

Incorporated by reference in its entirety herein is a computer-readablenucleotide/amino acid sequence listing submitted concurrently herewithand identified as follows: One 86,678 Byte ASCII (Text) file named“338844_0640_30A_US_ST25.TXT,” created on Jun. 25, 2021

BACKGROUND OF THE INVENTION

Excess intake of nutritive sweeteners has long been associated withdiet-related health issues, such as obesity, heart disease, metabolicdisorders and dental problems. Accordingly, consumers are increasinglylooking for ways to decrease the amount of nutritive sweeteners in theirdiets. Manufacturers are responding to this demand by seeking to developreplacements for nutritive sweeteners that are better able to mimic thedesirable taste and functional properties of the nutritive sweeteners.

Zero or low-calorie sweeteners derived from, preferably, natural sourcesare desired to limit the negative effects of high sugar consumption(e.g., diabetes and obesity, among others). Commonly-known zero orlow-calorie sweeteners include aspartame, acesulfame potassium, luo hanguo (monk) fruit extract, neotame, saccharin, stevia and sucralose.However, these sweeteners have taste defects such as bitterness.

A truffle is the subterranean fruiting body of some ascomycete fungiincluding genera which belong to the class Pezizomycetes orderPezizales. Truffles are ectomycorrhizal fungi and are therefore usuallyfound in close association with tree roots.

So far there are seven known sweet and taste-modifying proteins, namelybrazzein, thaumatin, monellin, curculin, mabinlin, miraculin andpentadin. The key residues on the protein surface responsible forbiological activity have not yet been identified with certainty for anyof these proteins. Monellin was found to be 100,000 times sweeter thansucrose on a molar basis, followed by brazzein and thaumatin which are500 times and 3000 times sweeter than sucrose, respectively, on a grambasis. All of these proteins have been isolated from plants that grow intropical rainforests. Although most of them share no sequence homologyor structural similarity, thaumatin shares extensive similarity at theprotein sequence level with certain non-sweet proteins found in otherplants. No sweet-taste modifying proteins are known from fungi.

There remains a need in the art to produce new low or zero caloriesweeteners with improved tastes from natural sources. There remains aneed in the art to economically produce such sweetening compositionsfrom potential sources of the same, particularly from ascomycetes fungalspecies.

SUMMARY OF THE INVENTION

The invention relates to newly identified fungally-derived sweetproteins, and to the genes and cDNA encoding said proteins, also calledMYD/Myd herein. More particularly, the invention relates to newlyidentified sweet-tasting proteins, to the genes and cDNA encoding saidproteins, and to methods of using such proteins, genes, and cDNA in themodulation of the taste of foods. The invention provides in particular aDNA sequence encoding a novel sweet protein identified herein as MYD andthe corresponding polypeptide Myd1 (also referred to as mycodulcein).Myd1 is the first sweet-tasting protein identified from fungi. Myd1reduces the sourness, bitterness or astringency of foods and drinks andadditionally Myd1 has an activity to enhance the taste of foods anddrinks, namely a taste-modifying activity.

The invention provides a polynucleotide (e.g., isolated polynucleotide)encoding a polypeptide having sweet-taste modulation activity, whereinthe polynucleotide sequence encodes a polypeptide selected from thegroup consisting of (a) a polypeptide sequence selected from the groupconsisting of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ IDNO:17, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, and SEQID NO:75; (b) a polypeptide having at least 80% sequence identity to thepolypeptide sequence selected from the group consisting of SEQ ID NO:3,SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO: 11, SEQ ID NO:12, SEQID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17;and (c) a polypeptide sequence modified from the polypeptide sequenceselected from the group consisting of SEQ ID NO:3, SEQ ID NO:8, SEQ IDNO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ IDNO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17 by deletion,insertion, substitution, or addition of no more than 24 amino acids,wherein the polypeptide encoding a polypeptide having sweet-tastemodulation activity is not the polypeptide of SEQ ID NO:3.

The invention also provides a polynucleotide (e.g., isolatedpolynucleotide) selected from the group consisting of: (a) apolynucleotide comprising the nucleic acid sequence set forth in SEQ IDNO:2 with at least one substitution modification; (b) a polynucleotidecomprising a nucleic acid sequence having at least 90% sequence identityto the nucleic acid sequence set forth in SEQ ID NO:2, wherein thepolynucleotide is not the polynucleotide of SEQ ID NO:2, and (c) apolynucleotide comprising (i) the nucleic acid sequence set forth in SEQID NO:2 and (ii) a nucleotide sequence encoding a histidine tag, whereinthe polynucleotide encodes a polypeptide having sweet-taste modulationactivity.

In one aspect, the polypeptide sequence of the polypeptide havingsweet-taste modulation activity comprises SEQ ID NO:71, SEQ ID NO:72,SEQ ID NO:73, SEQ ID NO:74, or SEQ ID NO:75. In particular, thepolypeptide sequence of the polypeptide having sweet-taste modulationactivity comprises SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 30, SEQ IDNO: 38, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 50, SEQID NO: 52, SEQ ID NO: 54, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62,SEQ ID NO: 64, SEQ ID NO; 66, or SEQ ID NO: 68.

In a particular aspect, the polynucleotide comprises the nucleic acidsequence of SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 29, SEQ ID NO: 37,SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 49, SEQ ID NO:51, SEQ ID NO: 53, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ IDNO; 63, SEQ ID NO: 65, or SEQ ID NO: 67.

The polynucleotide encoding the polypeptide having sweet-tastemodulation activity is optionally operably linked to a heterologousregulatory element. Additionally or alternatively, the polynucleotidesequence further encodes a protein tag or label. The protein tag isoptionally an affinity tag, The protein is optionally a histidine tag.

In one aspect, the polynucleotide comprises SEQ ID NO: 20, whichcorresponds to the coding sequence for His tagged mycodulcein in E. coli(wherein residues 364-381 correspond to an optional His tag sequence).SEQ ID NO: 20 is codon-optimized for expression in E. coli. In anotheraspect, the polynucleotide comprises SEQ ID NO: 22, which corresponds tothe coding sequence for His tagged mycodulcein in S. cerevisiae (whereinresidues 364-381 correspond to an optional His tag sequence). SEQ ID NO:22 is codon-optimized for expression in S. cerevisiae. The correspondingpolypeptide of SEQ ID NO: 21 corresponds to the His-tagged mycodulceinprotein (wherein residues 122-127 correspond to the optional His tagsequence), which polypeptide sequence is the same for expression in E.coli and S. cerevisiae.

An expression cassette comprising the polynucleotide and a vectorcomprising the polynucleotide, as well as a host cell transformed withthe vector is provided. A method of producing a protein havingsweet-taste modulation activity, comprising culturing the host cell in amedium under conditions that result in producing the protein havingsweet-taste modulation activity also is provided.

The invention includes a polypeptide (e.g., isolated polypeptide)comprising a polypeptide sequence having at least 80% sequence identityto a polypeptide sequence selected from the group consisting of SEQ IDNO:3, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ IDNO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQID NO:17; (a) wherein the polypeptide contains at least one substitutionor modification relative to the polypeptide sequence selected from thegroup consisting of SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10,SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15,SEQ ID NO:16, and SEQ ID NO:17, wherein the polypeptide is not thepolypeptide of SEQ ID NO:3, or (b) wherein the polypeptide furthercomprises a protein tag, particularly a histidine tag, and wherein thepolypeptide has sweet-taste modulation activity.

In one aspect, the polypeptide (e.g., isolated polypeptide) comprises apolypeptide sequence selected from the group consisting of SEQ ID NO:71,SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, and SEQ ID NO:75, wherein thepolypeptide optionally is not SEQ ID NO: 3. In particular, thepolypeptide comprises the amino acid sequence of SEQ ID NO: 24, SEQ IDNO: 26, SEQ ID NO: 30, SEQ ID NO: 38, SEQ ID NO: 42, SEQ ID NO: 44, SEQID NO: 46, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 58,SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO; 66, or SEQ IDNO: 68.

The invention provides a composition, comprising a combination of (a) aproduct for oral administration, wherein the product is notMattirolomyces terfezioides truffle, and (b) a sweetening compositioncomprising the polypeptide, wherein the combination has enhanced sweettaste compared to the product for oral administration. In one aspect,the polypeptide comprises an amino acid sequence having at least 80%sequence identity to a polypeptide selected from the group consisting ofSEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ IDNO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ IDNO:16, and SEQ ID NO:17. In another aspect, the polypeptide has apolypeptide sequence having at least 80% sequence identity to apolypeptide sequence selected from the group consisting of SEQ ID NO:3,SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17;(a) wherein the polypeptide contains at least one substitutionmodification relative to the polypeptide sequence selected from thegroup consisting of SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10,SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15,SEQ ID NO:16, and SEQ ID NO:17, wherein the polypeptide is not thepolypeptide of SEQ ID NO:3, or (b) wherein the polypeptide furthercomprises a histidine tag, wherein the polypeptide has sweet-tastemodulation activity. For example, the polypeptide in the sweeteningcomposition comprises the amino acid sequence of SEQ ID NO:71, SEQ IDNO:72, SEQ ID NO:73, SEQ ID NO:74, or SEQ ID NO:75 and optionally doesnot consist of SEQ ID NO:3. In particular, the polypeptide comprises theamino acid sequence of SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 30, SEQID NO: 38, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 50,SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO:62, SEQ ID NO: 64, SEQ ID NO; 66, or SEQ ID NO: 68.

In one aspect, the invention provides a sweetening compositioncomprising a polypeptide comprises an amino acid sequence having atleast 80% sequence identity to a polypeptide selected from the groupconsisting of SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ IDNO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ IDNO:15, SEQ ID NO:16, and SEQ ID NO:17, wherein the polypeptide is apolypeptide other than the polypeptide of the amino acid sequence of SEQID NO:3.

The invention provides a method for modulating the taste of a productfor oral administration, comprising combining the product for oraladministration with an effective amount of a sweetening compositioncomprising the polypeptide, wherein the product for oral administrationis not Mattirolomyces terfezioides truffle, and wherein the combinationhas enhanced sweet taste compared to the product for oraladministration. In one aspect, the polypeptide comprises an amino acidsequence having at least 80% sequence identity to a polypeptide selectedfrom the group consisting of SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:8, SEQID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ IDNO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17. In another aspect,the polypeptide has a polypeptide sequence having at least 80% sequenceidentity to a polypeptide sequence selected from the group consisting ofSEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, andSEQ ID NO:17; (a) wherein the polypeptide contains at least onesubstitution modification relative to the polypeptide sequence selectedfrom the group consisting of SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, SEQID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ IDNO:15, SEQ ID NO:16, and SEQ ID NO:17, wherein the polypeptide is notthe polypeptide of SEQ ID NO:3, or (b) wherein the polypeptide furthercomprises a histidine tag, wherein the polypeptide has sweet-tastemodulation activity. For example, the polypeptide in the sweeteningcomposition comprises the amino acid sequence of SEQ ID NO:71, SEQ IDNO:72, SEQ ID NO:73, SEQ ID NO:74, or SEQ ID NO:75 and optionally doesnot consist of SEQ ID NO:3. In particular, the polypeptide comprises theamino acid sequence of SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 30, SEQID NO: 38, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 50,SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO:62, SEQ ID NO: 64, SEQ ID NO; 66, or SEQ ID NO: 68.

The product for oral administration can be a food, a beverage, a dietarysupplement composition, or a pharmaceutical composition. Examples offood products include, but are not limited to, baked goods; sweet bakeryproducts, pre-made sweet bakery mixes for preparing sweet bakeryproducts; pie fillings and other sweet fillings, gelatins and puddings;frozen desserts; yogurts; snack bars; bread products; pre-made breadmixes for preparing bread products; sauces, syrups and dressings; sweetspreads; confectionary products; and sweetened breakfast cereals.Examples of beverage product include but are not limited to carbonatedbeverages; non-carbonated beverages; and beverage concentrates.

The invention also provides a method of purifying a polypeptide havingsweet-taste modulation activity comprising (a) obtaining a compositioncomprising the polypeptide, and (b) purifying the composition viahydrophobic interaction chromatography (HIC) followed by size exclusionchromatography (SEC). In one aspect, the polypeptide comprises an aminoacid sequence having at least 80% sequence identity to a polypeptideselected from the group consisting of SEQ ID NO:3, SEQ ID NO:7, SEQ IDNO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ IDNO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO: 17. Inanother aspect, the polypeptide has a polypeptide sequence having atleast 80% sequence identity to a polypeptide sequence selected from thegroup consisting of SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10,SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15,SEQ ID NO:16, and SEQ ID NO: 17; (a) wherein the polypeptide contains atleast one substitution modification relative to the polypeptide sequenceselected from the group consisting of SEQ ID NO:3, SEQ ID NO:8, SEQ IDNO:9, SEQ ID NO:10, SEQ ID NO: 11, SEQ ID NO:12, SEQ ID NO:13, SEQ IDNO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17, wherein thepolypeptide is not the polypeptide of SEQ ID NO:3, or (b) wherein thepolypeptide further comprises a histidine tag, wherein the polypeptidehas sweet-taste modulation activity. For example, the polypeptidecomprises the amino acid sequence of SEQ ID NO:71, SEQ ID NO:72, SEQ IDNO:73, SEQ ID NO:74, or SEQ ID NO:75 and optionally does not consist ofSEQ ID NO:3. In particular, the polypeptide comprises the amino acidsequence of SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 30, SEQ ID NO: 38,SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 50, SEQ ID NO:52, SEQ ID NO: 54, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ IDNO: 64, SEQ ID NO; 66, or SEQ ID NO: 68.

Other aspects and embodiments of the invention will be apparent onreview of the figures, detailed description and non-limiting examplesherein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a predicted three dimensional structure of Myd1 based onsequence data using the PHYRE2.0 protein folding prediction tool.

FIG. 2 shows a Coomassie-stained SDS-PAGE gel of proteins obtained fromfractions of partially purified M. terfezioides gleba.

FIG. 3 shows a SDS-PAGE gel, Coomassie stained, of purification steps ofSEQ ID NO:21 expressed in E. coli. lane 1: molecular weight standards;lane 3, crude lysate; lane 4, flow through fraction from HisPur™ Ni-NTA;lane 5, wash 1; lane 6, wash 2; lane 7, wash 3; lane 8, elutionfraction.

FIG. 4 shows the concentration-response functions for sweet taste formycodulcein, aspartame, thaumatin, rebaudioside A. Data are plotted asthe proportion (p) of responses occurring on the 200 mMsucrose-associated (“sweet”) target. Each data point in the curves formycodulcein, aspartame, thaumatin, rebaudioside A was calculated as theaverage across 32 replicates and averaged across 16 replicates for thesucrose curve; error bars are SEM. Points for water and sucrose controlswere similarly calculated as the average across 128 and 64 replicates,respectively. Curves were fit by non-linear regression.

FIG. 5A shows a comparison of mycodulcein predicted tertiary structureto the known crystal structures of thaumatin (PDB: 1RQW), monellin (PDB:209U), brazzein (PDB: 1BRZ), and chicken egg white lysozyme (PDB: 1LSN)(protein data base), showing that mycodulcein has predicted tertiarystructure similarity to other known sweet proteins, all containingantiparallel beta-sheets with an alpha-helix parallel to the betasheets.

FIG. 5B shows a representation of SEQ ID NO:3 predicted secondarystructure superimposed on putative secondary structure motifs andlocation of point mutations within each motif.

FIG. 6 shows results of mutant his-tagged mycodulcein compared with eachother and with his-tagged non-mutant mycodulcein, equalized to equalprotein concentration as measured by ELISA, for sweetness intensity,time of onset of sweetness perception, and for duration of sweetnessperception.

FIG. 7A shows SDS-PAGE analysis, Coomassie stain, of the eluted fractionfrom Capto MMC. M: protein marker; lane 1: eluted fraction, showing lowpurity after cation exchange. Arrow (1) indicates mycodulcein band.

FIG. 7B shows SDS-PAGE analysis, Coomassie stain, two eluted fractionscollected during the gradient elution from the HiScreen Capto Butylcolumn analyzed on SDS-PAGE. Lane 1 shows eluted fraction 1 notcontaining mycodulcein and Lane 2 shows eluted mycodulcein. The purityof the eluted fraction was determined by GelAnalyzer to be ˜86%. Arrow(2) indicates mycodulcein band.

FIG. 7C shows SDS-PAGE analysis, Coomassie stain, the eluted proteinfrom the HIC column after chromatographing on HiPrep 26/60 SephacrylS-200. Lane 1 shows purified his-tag mycodulcein and Lane 2 showspurified native mycodulcein. The purity of the eluted fraction wasdetermined by GelAnalyzer to be ˜98%. Arrow (3) indicates mycodulceinband.

DETAILED DESCRIPTION OF THE INVENTION

The invention thus provides isolated nucleic acid molecules encodingproteins which are capable of modulating sweet taste, and thepolypeptides they encode. As described herein, the polypeptides of theinvention mediate sweet taste perception, either alone, or incombination with food, beverage, dietary supplement, or pharmaceuticals.The invention also provides isolated polypeptides that are capable ofmodifying sweet taste, and compositions of same together with foods,beverages, dietary supplements, or pharmaceutical compositions, with theresultant combinations having a sweet taste. The present invention alsoprovides methods for modifying the sweet taste of foods, beverages,dietary supplements, or pharmaceutical compositions by using theisolated polynucleotides and polypeptides of the invention.

In one aspect of the invention, provided is a newly identified fungalsweet protein termed Myd1 herein. The term “Myd polypeptides” is usedherein to identify any of the polypeptides according to the presentinvention which e.g. have at least 80% sequence identity to SEQ ID NO:3and also have sweet-taste modifying activity. Myd polypeptides alsoinclude peptides SEQ ID NO:8 through SEQ ID NO:17 with sweet-tastemodifying activity. A sweet-tasting partially purified extract of M.terfezioides gleba was subjected to de novo amino acid sequencing toidentify a 20-mer N-terminal sequence (SEQ ID NO:4). The Myd1 codingsequence (putatively derived from the MYD gene) was identified after thewhole transcriptome of the M. terfeziodes gleba was de novo assembledusing RNAseq reads. Screening the M. terfeziodes whole transcriptomeusing the 20-mer N-terminus sequences identified a transcript predictedto encode a protein with 100% identity at the N-terminus. The identifiedtranscript is predicted to encode a 121 amino acid protein. This methodidentified SEQ ID NO:1. Start and stop codons were identified in thetranscript to identify putative coding sequence SEQ ID NO:2. SEQ ID NO:3is the putative protein, a 121 amino acid protein. Identity between thepredicted protein SEQ ID NO:3, and other protein sequences in GENBANKwere 31% or less. The coding sequences for native mycodulcein, whichhave been codon-optimized for expression in E. coli and Saccharomycescerevisiae correspond to the nucleic acid sequences of SEQ ID NO: 20 andSEQ ID NO:22, respectively (which encode the amino acid sequence of SEQID NO: 3 with an optional 6 residue histidine tag, i.e., the amino acidsequence of SEQ ID NO: 21).

In an aspect, the “Myd polypeptides” herein have sweet-taste modifyingactivity of at least 10% or higher (e.g., 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%. 90%, 95%, or higher)compared to the naturally occurring Myd polypeptide isolated from M.terfeziodes gleba by extraction. In other aspects, the “Mydpolypeptides” herein have sweet-taste modifying activity of at least 50%or higher compared to the naturally occurring Myd polypeptide isolatedfrom M. terfeziodes gleba by extraction. In an aspect, the “Mydpolypeptides” herein have sweet-taste modifying activity of at least 80%or higher compared to the naturally occurring Myd polypeptide isolatedfrom M. terfeziodes gleba by extraction. Sweet-taste modifying activitycan be measured by any comparative method known in the art and inparticular by any method as described in the examples herein and morespecifically employing a method using a sensory panel as describedherein.

While not wishing to be bound to any particular theory, Myd1 is believedto be involved in sweet taste activation e.g., is an agonist of taste 1receptor member 2 (T1R2) and/or taste 1 receptor member 3 (T1R3).However, Myd1 may agonize other taste receptors, such as bitter, umami,sour and salty. Isolated or purified Myd polypeptides can then be usedin the food and pharmaceutical industries to customize taste, e.g., tomodulate the sweet tastes of foods or drugs.

In a first aspect, the present invention comprises a polynucleotide(e.g., isolated polynucleotide) encoding a polypeptide havingsweet-taste modulation activity, wherein the polypeptide sequencecomprises, consists essentially of, or consists of a polypeptideselected from the group consisting of: (a) the amino acid sequence setforth in SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ IDNO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ IDNO:16, or SEQ ID NO:17; (b) an amino acid sequence having at least 80%(e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least85%, at least 86%, at least 87%, at least 88%, at least 89%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequenceidentity to the amino acid sequence set forth in SEQ ID NO:3, SEQ IDNO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ IDNO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, or SEQ ID NO:17; and(c) an amino acid sequence modified from the amino acid sequence setforth in SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ IDNO:16, or SEQ ID NO:17 by deletion, insertion, substitution, or additionof no more than 24 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, and any ranges thereof) aminoacids. In some embodiments, the amino acid sequence of the polypeptidehaving sweet-taste modulation activity is the amino acid sequence setforth in SEQ ID NO:3, an amino acid sequence having at least 80%sequence identity to SEQ ID NO:3, or an amino acid modified from theamino acid sequence SEQ ID NO:3 by deletion, insertion, substitution, oraddition of no more than 24 amino acids.

In a particular aspect, the invention provides a polynucleotide (e.g.,isolated polynucleotide) encoding a polypeptide having sweet-tastemodulation activity, wherein the polynucleotide sequence encodes apolypeptide selected from the group consisting of: (a) a polypeptidesequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO: 11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14,SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17; (b) a polypeptide havingat least 80% sequence identity to the polypeptide sequence selected fromthe group consisting of SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, SEQ IDNO:10, SEQ ID NO: 11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ IDNO:15, SEQ ID NO:16, and SEQ ID NO:17; and (c) a polypeptide sequencemodified from the polypeptide sequence selected from the groupconsisting of SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ IDNO:16, and SEQ ID NO:17 by deletion, insertion, substitution, oraddition of no more than 24 amino acids, wherein the isolatedpolypeptide encoding a polypeptide having sweet-taste modulationactivity is not the polypeptide of SEQ ID NO:3.

In another aspect, the present invention includes a polynucleotide(e.g., isolated polynucleotide) wherein the polynucleotide is selectedfrom the group consisting of: (a) a polynucleotide comprising thenucleic acid sequence set forth in SEQ ID NO: 2, optionally containingat least one (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,72, and ranges of any of these values) modification (e.g., deletion,insertion, substitution, or addition; and (b) a polynucleotidecomprising a nucleic acid sequence having at least 80% (e.g., at least81%, at least 82%, at least 83%, at least 84%, at least 85%, at least86%, at least 87%, at least 88%, at least 89%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99%) sequence identity tothe nucleic acid sequence set forth in SEQ ID NO:2, optionally whereinthe polynucleotide is not the polynucleotide of SEQ ID NO:2. In anembodiment, the polynucleotide encodes a polypeptide having sweet-tastemodulation activity. In an embodiment, the amino acid sequence of thepolypeptide having sweet-taste modulation activity is the amino acidsequence set forth in SEQ ID NO:3.

In one aspect, the invention provides a polynucleotide (e.g., isolatedpolynucleotide) wherein the polynucleotide sequence comprises, consistsessentially of, or consists of a polynucleotide selected from the groupconsisting of: (a) a polynucleotide comprising the nucleic acid sequenceset forth in SEQ ID NO:2 with at least one (e.g., 2, 3, 4, 5, 6, 7, 8,9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,64, 65, 66, 67, 68, 69, 70, 71, 72, and ranges of any of these values)substitution modification; (b) a polynucleotide comprising a nucleicacid sequence having at least 90% (at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, or at least 99%) sequence identity to the nucleic acid sequence setforth in SEQ ID NO:2, wherein the polynucleotide optionally is not thepolynucleotide of SEQ ID NO:2, and (c) a polynucleotide comprising (i)the nucleic acid sequence set forth in SEQ ID NO:2 and (ii) a nucleotidesequence encoding a histidine tag, wherein the polynucleotide encodes apolypeptide having sweet-taste modulation activity.

The polynucleotide encoding the Myd of the present invention can be inthe form of a single-stranded or double-stranded DNA, RNA or anartificial nucleic acid, or can be a cDNA or a chemically synthesizedDNA which does not comprise any intron.

The term “nucleic acid” or “nucleic acid sequence” refers to adeoxy-ribonucleotide or ribonucleotide oligonucleotide in either single-or double-stranded form. The term encompasses nucleic acids, i.e.,oligonucleotides, containing known analogs of natural nucleotides. Theterm also encompasses nucleic-acid-like structures with syntheticbackbones (see e.g., Oligonucleotides and Analogues, a PracticalApproach, ed. F. Eckstein, Oxford Univ. Press (1991); AntisenseStrategies, Annals of the N.Y. Academy of Sciences, Vol. 600, Eds.Baserga et al. (NYAS 1992); Milligan J. Med. Chem. 36:1923-1937 (1993);Antisense Research and Applications (1993, CRC Press), WO 97/03211; WO96/39154; Mata, Toxicol. Appl. Pharmacol. 144:189-197 (1997);Strauss-Soukup, Biochemistry 36:8692-8698 (1997); Samstag, AntisenseNucleic Acid Drug Dev, 6:153-156 (1996)).

Unless otherwise indicated, a particular nucleic acid sequence alsoimplicitly encompasses conservatively modified variants thereof (e.g.,degenerate codon substitutions) and complementary sequences, as well asthe sequence explicitly indicated. Specifically, degenerate codonsubstitutions may be achieved by generating, e.g., sequences in whichthe third position of one or more selected codons is substituted withmixed-base and/or deoxyinosine residues (Batzer et al., Nucleic AcidRes., 19:5081 (1991); Ohtsuka et al., J. Biol. Chem., 260:2605-2608(1985); Rossolini et al., Mol. Cell. Probes, 8:91-98 (1994)). The termnucleic acid is used interchangeably with gene, cDNA, mRNA,oligonucleotide, and polynucleotide.

The invention also provides an expression cassette comprising thepolynucleotide encoding a Myd polypeptide and a host cell transformedwith the vector.

In another aspect, the invention provides a polypeptide (e.g., anisolated polypeptide) comprising, consisting essentially of, orconsisting of a polypeptide sequence having at least 80% (e.g., at least81%, at least 82%, at least 83%, at least 84%, at least 85%, at least86%, at least 87%, at least 88%, at least 89%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99%) sequence identity to apolypeptide sequence selected from the group consisting of SEQ ID NO:3,SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO: 11, SEQ ID NO:12, SEQID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17,wherein the polypeptide optionally contains at least one (e.g., 2, 3, 4,5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, and ranges of any ofthese values) modification (e.g., deletion, insertion, substitution, oraddition), wherein the polypeptide optionally further comprises ahistidine tag, and wherein the polypeptide has sweet-taste modulationactivity. The term “consisting essentially of” allows for the inclusionof components that are not essential to the function or activity of theproduct and do not materially affect the function or activity, such ananti-caking agent, filler, stabilizer (e.g., thermal stabilizer), andbulking agent (e.g., maltodextrose, gum acacia and the like).

The polypeptide comprises SEQ ID NO: 3 or at least 80% sequence identityto SEQ ID NO: 3. The polypeptide comprises SEQ ID NO: 8 or at least 80%sequence identity to SEQ ID NO: 8. The polypeptide comprises SEQ ID NO:9 or at least 80% sequence identity to SEQ ID NO: 9. The polypeptidecomprises SEQ ID NO: 10 or at least 80% sequence identity to SEQ ID NO:10. The polypeptide comprises SEQ ID NO: 11 or at least 80% sequenceidentity to SEQ ID NO: 11. The polypeptide comprises SEQ ID NO: 12 or atleast 80% sequence identity to SEQ ID NO: 12. The polypeptide comprisesSEQ ID NO: 13 or at least 80% sequence identity to SEQ ID NO: 13. Thepolypeptide comprises SEQ ID NO: 14 or at least 80% sequence identity toSEQ ID NO: 14. The polypeptide comprises SEQ ID NO: 15 or at least 80%sequence identity to SEQ ID NO: 15. The polypeptide comprises SEQ ID NO:16 or at least 80% sequence identity to SEQ ID NO: 16. The polypeptidecomprises SEQ ID NO: 17 or at least 80% sequence identity to SEQ ID NO:17.

In one embodiment, the polypeptide sequence is selected from the groupconsisting of SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ IDNO:16, and SEQ ID NO:17. In one embodiment, the polypeptide is not thepolypeptide of SEQ ID NO: 3.

In one aspect, the polypeptide comprises amino acid residues 1-11,17-32, 39, 40, 45-67, 73-100, and 110-121 of SEQ ID NO:3. In one aspect,the polypeptide comprises amino acid residues 1-11, 17-32, 39, 40,45-67, 73-100, and 110-121 of SEQ ID NO:3, but the polypeptide is notthe polypeptide having the amino acid sequence of SEQ ID NO:3.

In another aspect, the polypeptide comprises amino acid residues 1-121of SEQ ID NO:3, wherein in amino acid residues 12-16, 33-38, 41-44,68-72, or 101-109 of the polypeptide sequence there is at least 1 (e.g.,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, or any range of values thereof) amino acid substitution,addition, insertion, or deletion compared to SEQ ID NO:3 in the listedresidues. In another aspect, the polypeptide sequence of the polypeptidehaving sweet-taste modulation activity encoded by the polynucleotide isa polypeptide comprising amino acid residues 1-121 of SEQ ID NO:3,wherein in amino acid residues 12-16, 33-38, 41-44, 68-72, or 101-109 ofthe polypeptide sequence there is at least 1 amino acid substitution,addition, insertion, or deletion compared to SEQ ID NO:3 in the listedresidues and wherein the polypeptide has at least 80% sequence identitywith SEQ ID NO:3.

In another aspect, the present invention includes a recombinantpolypeptide having sweet modulation activity comprising, consistingessentially of, or consisting of an amino acid sequence having at least80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%, atleast 85%, at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%)sequence identity to SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, SEQ IDNO:10, SEQ ID NO: 11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ IDNO:15, SEQ ID NO:16, or SEQ ID NO:17 fused to a heterologous signalpeptide or transit peptide. The term “consisting essentially of” allowsfor the inclusion of components that are not essential to the functionor activity of the product and do not materially affect the function oractivity, such an anti-caking agent, filler, stabilizer (e.g., thermalstabilizer), and bulking agent (e.g., maltodextrose, gum acacia and thelike).

In another aspect, the present invention includes a polypeptide havingsweet modulation activity comprising, consisting essentially of, orconsisting of an amino acid having at least 80% (e.g., at least 81%, atleast 82%, at least 83%, at least 84%, at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%) sequence identity to SEQ IDNO:3 (or, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ IDNO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, or SEQ IDNO:17), and containing at least one substitution modification relativeto SEQ ID NO:3 (or, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ IDNO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ IDNO:16, or SEQ ID NO:17). In some aspects, a polypeptide of the inventioncomprises 1 to 24 amino acid substitutions at position 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 42, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 56, 57, 58, 59, 60, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,84, 85 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101,102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115,116, 117, 118, 119, 120, or 121, compared to the corresponding aminoacid of SEQ ID NO:3. The term “consisting essentially of” allows for theinclusion of components that are not essential to the function oractivity of the product and do not materially affect the function oractivity, such an anti-caking agent, filler, stabilizer (e.g., thermalstabilizer), and bulking agent (e.g., maltodextrose, gum acacia and thelike).

In a particular aspect, the polypeptide sequence of the polypeptidehaving sweet-taste modulation activity that is encoded by thepolynucleotide comprises the amino acid sequence of SEQ ID NO: 71, SEQID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, or SEQ ID NO: 75.

SEQ ID NO: 71 (consensus sequence 1) corresponds to SEQ ID NO: 3 exceptthat positions 3, 11-16, 26, 33, 34, 36-38, 41-43, 51, 57, 66, 68-72,85, 86, 89, 97, 101-110, 117, and 120 are any amino acid. The inventionprovides a polypeptide comprising the amino acid sequence of SEQ ID NO:71.

SEQ ID NO: 72 (consensus sequence 2) corresponds to SEQ ID NO: 3 exceptthat positions 3, 11-16, 26, 33, 37, 38, 41, 43, 51, 57, 66, 68-70, 72,85, 86, 89, 97, 101-103, 105-110, 117, and 120 are any amino acid (i.e.,the prolines at positions 34, 36, 42, 71, and 104 of SEQ ID NO: 3 aremaintained). The invention provides a polypeptide comprising the aminoacid sequence of SEQ ID NO: 72.

SEQ ID NO: 73 (consensus sequence 3) and SEQ ID NO: 74 (consensussequence 4) correspond to SEQ ID NO: 3 except that positions 3, 11-16,26, 33, 37, 38, 41, 43, 51, 57, 66, 68-70, 72, 85, 86, 89, 97, 101-103,105-110, 117, and 120 can include conservative modifications asdescribed herein. The invention provides a polypeptide comprising theamino acid sequence of SEQ ID NO: 73. The invention provides apolypeptide comprising the amino acid sequence of SEQ ID NO: 74.

SEQ ID NO: 75 (consensus sequence 5) corresponds to SEQ ID NO: 3 exceptthat positions 3, 11, 26, 51, 57, 66, 69, 85, 86, 89, 97, 103, 106, 110,117, and 120 can include conservative modifications as described herein.The invention provides a polypeptide comprising the amino acid sequenceof SEQ ID NO: 75.

In particular aspect, the polypeptide sequence of the polypeptide havingsweet-taste modulation activity comprises SEQ ID NO: 3 with one or moremodifications (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,and 16) at residues 3, 11, 26, 51, 57, 66, 69, 85, 86, 89, 97, 103, 106,110, 117, and 120 of SEQ ID NO: 3. Exemplary modifications relative toSEQ ID NO: 3 (for the polypeptide) are set forth herein (see Example 8;Table 3). For example, the polypeptide sequence of the polypeptidehaving sweet-taste modulation activity comprises SEQ ID NO: 24, SEQ IDNO: 26, SEQ ID NO: 30, SEQ ID NO: 38, SEQ ID NO: 42, SEQ ID NO: 44, SEQID NO: 46, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 58,SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO; 64, SEQ ID NO: 66, or SEQ IDNO: 68 or an amino acid sequence with at least 80% (e.g., 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99%) sequence identity to SEQ ID NO: 24, SEQ ID NO: 26, SEQ IDNO: 30, SEQ ID NO: 38, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 58, SEQ ID NO: 60,SEQ ID NO: 62, SEQ ID NO; 64, SEQ ID NO: 66, or SEQ ID NO: 68.

The polypeptide sequence of the polypeptide having sweet-tastemodulation activity comprises SEQ ID NO: 24 (D3E) or at least 80%sequence identity to SEQ ID NO: 24. The polypeptide sequence of thepolypeptide having sweet-taste modulation activity comprises SEQ ID NO:26 (K11R) or at least 80% sequence identity to SEQ ID NO: 26. Thepolypeptide sequence of the polypeptide having sweet-taste modulationactivity comprises SEQ ID NO: 30 (K26R) or at least 80% sequenceidentity to SEQ ID NO: 30. The polypeptide sequence of the polypeptidehaving sweet-taste modulation activity comprises SEQ ID NO: 38 (K51R) orat least 80% sequence identity to SEQ ID NO: 38. The polypeptidesequence of the polypeptide having sweet-taste modulation activitycomprises SEQ ID NO: 42 (R57K) or at least 80% sequence identity to SEQID NO: 42. The polypeptide sequence of the polypeptide havingsweet-taste modulation activity comprises SEQ ID NO: 44 (R66K) or atleast 80% sequence identity to SEQ ID NO: 44. The polypeptide sequenceof the polypeptide having sweet-taste modulation activity comprises SEQID NO: 46 (D69E) or at least 80% sequence identity to SEQ ID NO: 46. Thepolypeptide sequence of the polypeptide having sweet-taste modulationactivity comprises SEQ ID NO: 50 (D85E) or at least 80% sequenceidentity to SEQ ID NO: 50. The polypeptide sequence of the polypeptidehaving sweet-taste modulation activity comprises SEQ ID NO: 52 (E86D) orat least 80% sequence identity to SEQ ID NO: 52. The polypeptidesequence of the polypeptide having sweet-taste modulation activitycomprises SEQ ID NO: 54 (E89D) or at least 80% sequence identity to SEQID NO: 54. The polypeptide sequence of the polypeptide havingsweet-taste modulation activity comprises SEQ ID NO: 58 (D97E) or atleast 80% sequence identity to SEQ ID NO: 58. The polypeptide sequenceof the polypeptide having sweet-taste modulation activity comprises SEQID NO: 60 (K103R) or at least 80% sequence identity to SEQ ID NO: 60.The polypeptide sequence of the polypeptide having sweet-tastemodulation activity comprises SEQ ID NO: 62 (R106K) or at least 80%sequence identity to SEQ ID NO: 62. The polypeptide sequence of thepolypeptide having sweet-taste modulation activity comprises SEQ ID NO:64 (R110K) or at least 80% sequence identity to SEQ ID NO: 64. Thepolypeptide sequence of the polypeptide having sweet-taste modulationactivity comprises SEQ ID NO: 66 (E117D) or at least 80% sequenceidentity to SEQ ID NO: 66. The polypeptide sequence of the polypeptidehaving sweet-taste modulation activity comprises SEQ ID NO: 68 (K120R)or at least 80% sequence identity to SEQ ID NO: 68.

In another aspect, the polynucleotide encoding the polypeptide havingsweet-taste modulation activity comprises SEQ ID NO: 23, SEQ ID NO: 25,SEQ ID NO: 29, SEQ ID NO: 37, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO:45, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 57, SEQ IDNO: 59, SEQ ID NO: 61, SEQ ID NO; 63, SEQ ID NO: 65, or SEQ ID NO: 67 oran nucleic acid sequence with at least 80% (e.g., 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99%) sequence identity to SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 29,SEQ ID NO: 37, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO:49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 57, SEQ ID NO: 59, SEQ IDNO: 61, SEQ ID NO; 63, SEQ ID NO: 65, or SEQ ID NO: 67.

The polynucleotide comprises SEQ ID NO: 23 (corresponding to D3E) or atleast 80% sequence identity to SEQ ID NO: 23. The polynucleotidecomprises SEQ ID NO: 25 (corresponding to K11R) or at least 80% sequenceidentity to SEQ ID NO: 25. The polynucleotide comprises SEQ ID NO: 29(corresponding to K26R) or at least 80% sequence identity to SEQ ID NO:29. The polynucleotide comprises SEQ ID NO: 37 (corresponding to K51R)or at least 80% sequence identity to SEQ ID NO: 37. The polynucleotidecomprises SEQ ID NO: 41 (corresponding to R57K) or at least 80% sequenceidentity to SEQ ID NO: 41. The polynucleotide comprises SEQ ID NO: 43(corresponding to R66K) or at least 80% sequence identity to SEQ ID NO:43. The polynucleotide comprises SEQ ID NO: 45 (corresponding to D69E)or at least 80% sequence identity to SEQ ID NO: 45. The polynucleotidecomprises SEQ ID NO: 49 (D85E) or at least 80% sequence identity to SEQID NO: 49. The polynucleotide comprises SEQ ID NO: 51 (corresponding toE86D) or at least 80% sequence identity to SEQ ID NO: 51. Thepolynucleotide comprises SEQ ID NO: 53 (corresponding to E89D) or atleast 80% sequence identity to SEQ ID NO: 53. The polynucleotidecomprises SEQ ID NO: 57 (corresponding to D97E) or at least 80% sequenceidentity to SEQ ID NO: 57. The polynucleotide comprises SEQ ID NO: 59(corresponding to K103R) or at least 80% sequence identity to SEQ ID NO:59. The polynucleotide comprises SEQ ID NO: 61 (corresponding to R106K)or at least 80% sequence identity to SEQ ID NO: 61. The polynucleotidecomprises SEQ ID NO: 63 (R110K) or at least 80% sequence identity to SEQID NO: 63. The polynucleotide comprises SEQ ID NO: 65 (E117D) or atleast 80% sequence identity to SEQ ID NO: 65. The polynucleotidecomprises SEQ ID NO: 67 (K120R) or at least 80% sequence identity to SEQID NO: 67.

In one aspect, the polynucleotide comprises SEQ ID NO: 20, whichcorresponds to the coding sequence for His tagged mycodulcein in E. coli(wherein residues 364-381 correspond to an optional His tag sequence).SEQ ID NO: 20 is codon-optimized for expression in E. coli. In anotheraspect, the polynucleotide comprises SEQ ID NO: 22, which corresponds tothe coding sequence for His tagged mycodulcein in S. cerevisiae (whereinresidues 364-381 correspond to an optional His tag sequence). SEQ ID NO:22 is codon-optimized for expression in S. cerevisiae. The correspondingpolypeptide of SEQ ID NO: 21 corresponds to the His-tagged mycodulceinprotein (wherein residues 122-127 correspond to the optional His tagsequence), which polypeptide sequence is the same for expression in E.coli and S. cerevisiae. Therefore, the invention also provides apolypeptide comprising the amino acid sequence of SEQ ID NO: 21.

The invention also provides a polypeptide comprising the amino acidsequence of SEQ ID NO: 28, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36,SEQ ID NO: 40, SEQ ID NO: 48, or SEQ ID NO: 56 or an amino acid sequencewith at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identityto SEQ ID NO: 28, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ IDNO: 40, SEQ ID NO: 48, or SEQ ID NO: 56.

The polypeptide comprises SEQ ID NO: 28 (R20K) or at least 80% sequenceidentity to SEQ ID NO: 28. The polypeptide comprises SEQ ID NO: 32(E35D) or at least 80% sequence identity to SEQ ID NO: 32. Thepolypeptide comprises SEQ ID NO: 34 (K44R) or at least 80% sequenceidentity to SEQ ID NO: 34. The polypeptide comprises SEQ ID NO: 36(D46E) or at least 80% sequence identity to SEQ ID NO: 36. Thepolypeptide comprises SEQ ID NO: 40 (D52E) or at least 80% sequenceidentity to SEQ ID NO: 40. The polypeptide comprises SEQ ID NO: 48(R75K) or at least 80% sequence identity to SEQ ID NO: 48. Thepolypeptide comprises SEQ ID NO: 56 (D94E) or at least 80% sequenceidentity to SEQ ID NO: 56.

In another aspect, the polynucleotide comprises SEQ ID NO: 27, SEQ IDNO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:39, SEQ ID NO:47, or SEQ IDNO:55 or a nucleic acid sequence with at least 80% (e.g., 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99%) sequence identity to SEQ ID NO: 27, SEQ ID NO:31, SEQ IDNO:33, SEQ ID NO:35, SEQ ID NO:39, SEQ ID NO:47, or SEQ ID NO: 55.

The polynucleotide comprises SEQ ID NO: 27 (corresponding to R20K) or atleast 80% sequence identity to SEQ ID NO: 27. The polynucleotidecomprises SEQ ID NO: 31 (corresponding to E35D) or at least 80% sequenceidentity to SEQ ID NO: 31. The polynucleotide comprises SEQ ID NO: 33(corresponding to K44R) or at least 80% sequence identity to SEQ ID NO:33. The polynucleotide comprises SEQ ID NO: 35 (corresponding to D46E)or at least 80% sequence identity to SEQ ID NO: 35. The polynucleotidecomprises SEQ ID NO: 39 (corresponding to D52E) or at least 80% sequenceidentity to SEQ ID NO: 39. The polynucleotide comprises SEQ ID NO: 47(corresponding to R75K) or at least 80% sequence identity to SEQ ID NO:47. The polynucleotide comprises SEQ ID NO: 55 (corresponding to D94E)or at least 80% sequence identity to SEQ ID NO: 55.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers and non-naturally occurring amino acid polymers.

The polynucleotide or polypeptide can be naturally occurring ornon-naturally occurring (e.g., synthetic, recombinant, modified, and/orvariant products). In one aspect, the naturally occurring ornon-naturally occurring products are isolated or purified.

In one aspect, the term “isolated” encompasses products that have beenremoved from a biological environment (e.g., a cell, tissue, culturemedium, body fluid, etc.), or otherwise increased in purity to anydegree (e.g., isolated from a synthesis medium). Isolated products,thus, can be synthetic or naturally produced.

As used herein, the term “isolated,” when referring to a nucleic acid orpolypeptide refers to a state of purification or concentration differentthan that which occurs naturally. Any degree of purification orconcentration greater than that which occurs naturally, including (1)the purification from other naturally occurring associated structures orcompounds, or (2) the association with structures or compounds to whichit is not normally associated in the body are within the meaning of“isolated” as used herein. The nucleic acids or polypeptides describedherein may be isolated or otherwise associated with structures orcompounds to which they are not normally associated in nature, accordingto a variety of methods and processed known to those of skill in theart. In one embodiment, the polypeptides described herein contain atmost 5% (e.g., at most 4%, at most 3%, at most 2% at most 1%) by weightof other fungal proteins.

As used herein, “recombinant” refers to a polynucleotide synthesized orotherwise manipulated in vitro (e.g., “recombinant polynucleotide”), tomethods of using recombinant polynucleotides to produce gene products incells or other biological systems, or to a polypeptide (“recombinantprotein”) encoded by a recombinant polynucleotide. “Recombinant means”also encompass the ligation of nucleic acids having various codingregions or domains or promoter sequences from different sources into anexpression cassette or vector for expression of, e.g., inducible orconstitutive expression of a fusion protein comprising a translocationdomain of the invention and a nucleic acid sequence amplified using aprimer of the invention.

“Modified” or “variant” products refer to products (e.g.,polynucleotides or polypeptides) that have been altered from theoriginal (e.g., naturally occurring) structure. As described herein,variants encompass polynucleotides or polypeptides with one or morechanges to the nucleic acid or amino acid sequences, respectively.Changes includes modifications to the nucleic acid or amino acidsequence, including additions, deletions, insertions, and substitutions.Modified or variant products also can encompass disulfide bondformation, glycosylation, lipidation, acetylation, phosphorylation, orany other manipulation, such as conjugation with a labeling component,relative to the original structure.

As used herein, the terms “amplifying” and “amplification” refer to theuse of any suitable amplification methodology for generating ordetecting recombinant or naturally expressed nucleic acid, as describedin detail, below. For example, the invention provides methods andreagents (e.g., specific degenerate oligonucleotide primer pairs) foramplifying (e.g., by polymerase chain reaction, PCR) naturally expressed(e.g., genomic or mRNA) or recombinant (e.g., cDNA) nucleic acids of theinvention (e.g., taste stimulus-binding sequences of the invention) invivo or in vitro.

The term “library” means a preparation that is a mixture of differentnucleic acid or polypeptide molecules, such as the library ofrecombinantly generated Myd associated polynucleotides generated byamplification of nucleic acid with degenerate primer pairs, or anisolated collection of vectors that incorporate the amplifiedligand-binding domains, or a mixture of cells each randomly transfectedwith at least one vector encoding a MYD.

As used herein a “nucleic acid probe or oligonucleotide” is defined as anucleic acid capable of binding to a target nucleic acid ofcomplementary sequence through one or more types of chemical bonds,usually through complementary base pairing, usually through hydrogenbond formation. As used herein, a probe may include natural (i.e., A, G,C, or T) or modified bases (7-deazaguanosine, inosine, etc.). Inaddition, the bases in a probe may be joined by a linkage other than aphosphodiester bond, so long as it does not interfere withhybridization. Thus, for example, probes may be peptide nucleic acids inwhich the constituent bases are joined by peptide bonds rather thanphosphodiester linkages. It will be understood by one of skill in theart that probes may bind target sequences lacking completecomplementarity with the probe sequence depending upon the stringency ofthe hybridization conditions. The probes are optionally directly labeledas with isotopes, chromophores, lumiphores, chromogens, or indirectlylabeled such as with biotin to which a streptavidin complex may laterbind. By assaying for the presence or absence of the probe, one candetect the presence or absence of the select sequence or subsequence.

The term “heterologous” when used with reference to portions of anucleic acid indicates that the nucleic acid comprises two or moresubsequences that are not found in the same relationship to each otherin nature. For instance, the nucleic acid is typically recombinantlyproduced, having two or more sequences from unrelated genes arranged tomake a new functional nucleic acid, e.g., a promoter from one source anda coding region from another source. Similarly, a heterologous proteinindicates that the protein comprises two or more subsequences that arenot found in the same relationship to each other in nature (e.g., afusion protein).

A “promoter” is defined as an array of nucleic acid sequences thatdirect transcription of a nucleic acid. As used herein, a promoterincludes necessary nucleic acid sequences near the start site oftranscription, such as, in the case of a polymerase II type promoter, aTATA element. A promoter also optionally includes distal enhancer orrepressor elements, which can be located as much as several thousandbase pairs from the start site of transcription. A “constitutive”promoter is a promoter that is active under most environmental anddevelopmental conditions. An “inducible” promoter is a promoter that isactive under environmental or developmental regulation. The term“operably linked” refers to a functional linkage between a nucleic acidexpression control sequence (such as a promoter, or array oftranscription factor binding sites) and a second nucleic acid sequence,wherein the expression control sequence directs transcription of thenucleic acid corresponding to the second sequence.

The term “MYD family” can refer to polymorphic variants, includingnatural alleles, mutants, alleles, and interspecies homologs that encodepolypeptides that: (1) have at least about 35 to 50% amino acid sequenceidentity, optionally about 60, 75, 80, 85, 90, 95, 96, 97, 98, or 99%amino acid sequence identity to SEQ ID NO:3 over a window of about 25amino acids, optionally 50-100 amino acids.

The term “expression vector” or “expression cassette” refers to anyrecombinant expression system for the purpose of expressing a nucleicacid sequence of the invention in vitro or in vivo, constitutively orinducibly, in any cell, including prokaryotic, yeast, fungal, plant,insect or mammalian cell. The term includes linear or circularexpression systems. The term includes expression systems that remainepisomal or integrate into the host cell genome. The expression systemscan have the ability to self-replicate or not, i.e., drive onlytransient expression in a cell. The term includes recombinant expression“cassettes which contain only the minimum elements needed fortranscription of the recombinant nucleic acid.

By “host cell” is meant a cell that contains an expression vector andsupports the replication or expression of the expression vector. Hostcells may be prokaryotic cells such as E. coli, or eukaryotic cells suchas yeast, insect, amphibian, or mammalian cells such as CHO, HeLa,HEK-293, and the like, e.g., cultured cells, explants, and cells invivo.

In one embodiment, the host cell is selected from the group consistingof Escherichia coli, Klebsiella oxytoca, Anaerobiospirillumsucciniciproducens, Actinobacillus succinogenes, Mannheimiasucciniciproducens, Agrobacterium tumefaciens, Rhizobium etli, Bacillussubtilis, Corynebacterium glutamicum, Gluconobacter oxydans, Zymomonasmobilis, Lactococcus lactis, Lactobacillus plantarum, Streptomycescoelicolor, Clostridium acetobutylicum, Pseudomonas fluorescens,Pseudomonas putida, Saccharomyces cerevisiae, Schizosaccharomyces pombe,Kluyveromyces lactis, Kluyveromyces marxianus, Aspergillus terreus,Aspergillus niger, Pichia pastoris, Rhizopus arrhizus, Rhizopus oryzae,Yarrowia lipolytica, Candida albicans, Issatchenkia orientalis,Scheffersomyces stipitis, Yarrowia lipolytica, Ogataea polymorpha,Phaffia rhodozyma, Candida utilis, Arxula adeninivorans, Debaryomyceshansenii, Debaryomyces polymorphus, and Schwanniomyces occidentalis.

In another aspect, the host cell is selected from the group consistingof gram-positive no-spore forming bacteria, gram-positive spore formingbacteria, gram negative bacteria, yeast, and protists/algae.

Non-limiting examples of gram-positive no-spore forming bacteria includeBifidobacterium adolescentis, Bifidobacterium animalis, Bifidobacteriumbifidum, Bifidobacterium breve, Bifidobacterium longum, Carnobacteriumdivergens, Corynebacterium ammoniagenes, Corynebacterium glutamicum,Lactobacillus acidophilus, Lactobacillus amylolyticus, Lactobacillusamylovorus, Lactobacillus animalis, Lactobacillus alimentarius,Lactobacillus aviaries, Lactobacillus brevis, Lactobacillus buchneri,Lactobacillus casei, Lactobacillus cellobiosus, Lactobacilluscollinoides, Lactobacillus corynformis, Lactobacillus crispatus,Lactobacillus curvatus, Lactobacillus delbrueckii, Lactobacillusdextrinicus, Lactobacillus diolivorans, Lactobacillus farciminis,Lactobacillus fermentum, Lactobacillus gallinarum, Lactobacillusgasseri, Lactobacillus helveticus, Lactobacillus hilgardii,Lactobacillus johnsonii, Lactobacillus kefiranofaciens, Lactobacilluskefiri, Lactobacillus mucosae, Lactobacillus panis, Lactobacillusparacasei, Lactobacillus parafarraginis, Lactobacillus paraplantarum,Lactobacillus pentosus, Lactobacillus plantarum, Lactobacillus pontis,Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus sakei,Lactobacillus salivarius, Lactobacillus sanfranciscensis, Lactococcuslactis, Leuconostoc citreum, Leuconostoc lactis, Leuconostocmesenteroides, Leuconostoc pseudomesenteroides, Microbacterium imperial,Oenococcus oeni, Pasteuria nishizawae, Pediococcus acidilactic,Pediococcus parvulus, Pediococcus pentosaceus, Propionibacteriumacidipropioni, Propionibacterium freudenreichii, and Streptococcusthermophiles.

Non-limiting examples of gram-positive spore forming bacteria includeBacillus amyloliquefaciens, Bacillus atrophaeus, Bacillus circulans,Bacillus clausii, Bacillus coagulans, Bacillus flexus, Bacillusfusiformis, Bacillus lentus, Bacillus lichenformis, Bacillus megaterium,Bacillus mojavensis, Bacillus pumilus, Bacillus smithii, Bacillussubtilis, Bacillus vallismortis, Bacillus velezensis, Geobacillusstearothermophilus, Paenibacillus illinoisensis, and Parageobacillusthermoglucosidasius. Non-limiting examples of gram negative bacteriainclude Cupriavidus necator, Gluconobacter oxydans, Komagataeibactersucrofermentans, and Xanthomonas campestris.

Non-limiting examples of yeast include Candida cylindracea, Debaryomyceshansenii, Hanseniaspora uvarum, Kluyveromyces lactis, Kluyveromycesmarxianus, Komagataella pastoris, Komagataella phaffi, Lindnera jadinii,Ogataea angusta, Saccharomyces bayanus, Saccharomyces cerevisiae,Saccharomyces pastorianus, Schizosaccharomyces pombe, Wickerhamomycesanomalus, Xanthophyllomyces dendrorhous, Yarrowia lipolytica, andZygosaccharomyces rouxii.

Non-limiting examples of protists/algae include Aurantiochytriumlimacinum, Euglena gracilis, and Tetraselmis chuii.

The Myd proteins described herein also include “analogs,” or“conservative variants” and “mimetics” (“peptidomimetics”) withstructures and activity that substantially correspond to the exemplarysequences. Thus, the terms “conservative variant” or “analog” or“mimetic” refer to a polypeptide which has a modified amino acidsequence, such that the change(s) do not substantially alter thepolypeptide's (the conservative variant's) structure and/or activity, asdefined herein. These include conservatively modified variations of anamino acid sequence, i.e., amino acid substitutions, additions ordeletions of those residues that are not critical for protein activity,or substitution of amino acids with residues having similar properties(e.g., acidic, basic, positively or negatively charged, polar ornon-polar, etc.) such that the substitutions of even critical aminoacids does not substantially alter structure and/or activity.

More particularly, “conservatively modified variants” applies to bothamino acid and nucleic acid sequences. With respect to particularnucleic acid sequences, conservatively modified variants refers to thosenucleic acids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a large number of functionally identical nucleicacids encode any given protein.

For instance, the codons GCA, GCC, GCG and GCU all encode the amino acidalanine. Thus, at every position where an alanine is specified by acodon, the codon can be altered to any of the corresponding codonsdescribed without altering the encoded polypeptide.

Such nucleic acid variations are “silent variations,” which are onespecies of conservatively modified variations. Every nucleic acidsequence herein which encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of skill willrecognize that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and TGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Accordingly, each silent variation of a nucleic acidwhich encodes a polypeptide is implicit in each described sequence.

Conservative substitution tables providing functionally similar aminoacids are well known in the art. For example, one exemplary guideline toselect conservative substitutions includes (original residue followed byexemplary substitution): ala/gly or ser; arg/lys; asn/gln or his;asp/glu; cys/ser; gln/asn; gly/asp; gly/ala or pro; his/asn or gln;ile/leu or val; leu/ile or val; lys/arg or gln or glu; met/leu or tyr orile; phe/met or leu or tyr; ser/thr; thr/ser; trp/tyr; tyr/trp or phe;val/ile or leu. An alternative exemplary guideline uses the followingsix groups, each containing amino acids that are conservativesubstitutions for one another: 1) Alanine (A), Serine (S), Threonine(T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N),Glutamine (Q); 4) Arginine (R), Lysine (I); 5) Isoleucine (I), Leucine(L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y),Tryptophan (W); (see also, e.g., Creighton, Proteins, W.H. Freeman andCompany (1984); Schultz and Schimer, Principles of Protein Structure,Springer-Vrlag (1979)). Another alternative exemplary guidelines usesthe following six groups, where proline is unique: 1) Gly (G), Ala (A),Val (V), Leu (L), Ile (I); 2) Ser (S), Cys (C), Thr (T), Met (M); 3) Pro(P); 4) Phe (F), Tyr (Y), Try (W); 5) His (H), Lys (K), Arg (R); and 6)Asp (D), Glu (E), Gln (N). One of skill in the art will appreciate thatthe above-identified substitutions are not the only possibleconservative substitutions. For example, for some purposes, one mayregard all charged amino acids as conservative substitutions for eachother whether they are positive or negative. In addition, individualsubstitutions, deletions or additions that alter, add or delete a singleamino acid or a small percentage of amino acids in an encoded sequencecan also be considered “conservatively modified variations.” One ofskill in the art would be familiar with codon selection in a given hostthat is expressing the protein of interest.

The terms “mimetic” and “peptidomimetic” refer to a synthetic chemicalcompound that has substantially the same structural and/or functionalcharacteristics of the polypeptides, e.g., translocation domains,ligand-binding domains, or chimeric receptors of the invention. Themimetic can be either entirely composed of synthetic, non-naturalanalogs of amino acids, or may be a chimeric molecule of partly naturalpeptide amino acids and partly non-natural analogs of amino acids. Themimetic can also incorporate any amount of natural amino acidconservative substitutions as long as such substitutions also do notsubstantially alter the mimetic's structure and/or activity.

As with polypeptides of the invention which are conservative variants,routine experimentation will determine whether a mimetic is within thescope of the invention, i.e., that its structure and/or function is notsubstantially altered. Polypeptide mimetic compositions can contain anycombination of non-natural structural components, which are typicallyfrom three structural groups: a) residue linkage groups other than thenatural amide bond (“peptide bond”) linkages; b) non-natural residues inplace of naturally occurring amino acid residues; or c) residues whichinduce secondary structural mimicry, i.e., to induce or stabilize asecondary structure, e.g., a beta turn, gamma turn, beta sheet, alphahelix conformation, and the like. A polypeptide can be characterized asa mimetic when all or some of its residues are joined by chemical meansother than natural peptide bonds. Individual peptidomimetic residues canbe joined by peptide bonds, other chemical bonds or coupling means, suchas, e.g., glutaraldehyde, N-hydroxysuccinimide esters, bifunctionalmaleimides, N,N′-dicyclohexylcarbodiimide (DCC) orN,N′-diisopropylcarbodiimide (DIC). Linking groups that can be analternative to the traditional amide bond (“peptide bond”) linkagesinclude, e.g., ketomethylene (e.g., —C(O)—CH₂— for —C(O)—NH—),aminomethylene (CH₂—NH), ethylene, olefin (CH═CH), ether (CH₂—O),thioether (CH—S), tetrazole (CN₄), thiazole, retroamide, thioamide, orester (see, e.g., Spatola, Chemistry and Biochemistry of Amino Acids,Peptides and Proteins, Vol. 7, pp 267-357, “Peptide BackboneModifications,” Marcell Dekker, N.Y. (1983)). A polypeptide can also becharacterized as a mimetic by containing all or some non-naturalresidues in place of naturally occurring amino acid residues;non-natural residues are well described in the scientific and patentliterature. Phyre2 is a suite of tools available on the web to predictand analyze protein structure, function and mutations.

A protein folding analysis is performed using tools including PHYREProtein Homology/analogY Recognition Engine V 2.0 and JPred, a ProteinSecondary Structure Prediction server. These tools reveal that SEQ IDNO:3 predicts a β sheet-dominated globular-type protein with significantsections of β-sheet and some short sections of α-helix. Specifically,the Jpred tool predicts putative β sheet from about residues 5-11,16-19, 28-29, 39-40, 61-67, 71-77, and 98-101; and α-helix from aboutresidues 20-25, 45-55, 111-119 of SEQ ID NO:3; the PHYRE tool predicts βsheet from about residue 5 to 12, 17-32, 38-40, 45-57, 62-66, 73-81,98-104, and 112-116 and α-helix from about residue 84 to 89 and 116-118of SEQ ID NO:3. See FIG. 1.

Examples of conservatively modified variations of Myd1 protein structuremay be derived using homology-modelling algorithms: SWISS-MODEL,PHYRE2.0, and Jpred to identify a sequence-based consensus loop regions,as known in the art, see, e.g., Pechmann, S. & Frydman, J. Interplaybetween Chaperones and Protein Disorder Promotes the Evolution ofProtein Networks. PLoS Computational Biology 10, e1003674 (2014).

Alternative protein sequences with putative similar structure andfunction to Myd1 are provided herein as SEQ ID NO:8 through SEQ IDNO:17. To derive SEQ ID NO:8 to SEQ ID NO:17, the consensus loop regionwas chosen as sites of mutation as most insertions and deletions areusually found in regions between secondary structure elements, wherethey can be accommodated easier without major distortions in the overallfold of the protein. The core of this protein has a higher degree ofsequence conservation as was found within 4JOX.

Out of the 29 possible amino acid locations within the consensus loopregion, 12 amino acids were substituted using conservative replacements.If selected for mutation, the wild type amino acid was given an equalprobability among the conservative amino acid residues. (Gly can bereplaced with Ala, Cys, Asp, Glu, Arg with an equal 20% probability).

Specific regions of the MYD/Myd nucleotide and amino acid sequences maybe used to identify polymorphic variants, interspecies homologs, andalleles of Myd family members. This identification can be made in vitro,e.g., under stringent hybridization conditions or PCR (e.g., usingprimers encoding the Myd sequences identified herein), or by using thesequence information in a computer system for comparison with othernucleotide sequences. Different alleles of MYD genes within a singlespecies population will also be useful in determining whetherdifferences in allelic sequences correlate to differences in tasteperception between members of the population. Classical PCR-typeamplification and cloning techniques are useful for isolating orthologs,for example, where degenerate primers are sufficient for detectingrelated genes across species.

For instance, primers designed using the sequences disclosed herein canbe used can be used to amplify and clone MYD-related genes fromdifferent fungal genomes. In contrast, genes within a single speciesthat are related to MYD are best identified using sequence patternrecognition software to look for related sequences. Typically,identification of polymorphic variants and alleles of MYD family memberscan be made by comparing an amino acid sequence of about 25 amino acidsor more, e.g., 50-100 amino acids. Amino acid identity of approximatelyat least 35 to 50%, and optionally 60%, 70%, 75%, 80%, 85%, 90%, 95-99%,or above typically demonstrates that a protein is a polymorphic variant,interspecies homolog, or allele of a MYD family member. Sequencecomparison can be performed using any of the sequence comparisonalgorithms discussed below. Antibodies that bind specifically to Mydpolypeptides or a conserved region thereof can also be used to identifyalleles, interspecies homologs, and polymorphic variants.

Nucleotide and amino acid sequence information for MYD family membersmay also be used to construct models of sweet modulating polypeptides ina computer system and how they interact with sweet receptors andcomputer system models of same. Sweet taste receptors are composed of aheterodimer of taste 1 receptor member 2 (T1R2) and taste 1 receptormember 3 (T1R3). These models can be subsequently used to identifyvariants and mutations of Myd that can increase activation of sweetreceptors and identify more active versions of Myd.

Various conservative mutations and substitutions are envisioned to bewithin the scope of the invention. For instance, it would be within thelevel of skill in the art to perform amino acid substitutions usingknown protocols of recombinant gene technology including PCR, genecloning, site-directed mutagenesis of cDNA, transfection of host cells,and in-vitro transcription. The variants could then be screened fortaste receptor agonist functional activity.

In one embodiment, hybrid protein-coding sequences comprising nucleicacids encoding Myds fusion proteins may be constructed. These nucleicacid sequences can be operably linked to transcriptional ortranslational control elements, e.g., transcription and translationinitiation sequences, promoters and enhancers, transcription andtranslation terminators, polyadenylation sequences, and other sequencesuseful for transcribing DNA into RNA. Fusion proteins may includeC-terminal or N-terminal translocation sequences. Further, fusionproteins can comprise additional elements, e.g., for protein detection,purification, or other applications. Detection and purificationfacilitating domains include, e.g., metal chelating peptides such aspolyhistidine tracts, histidine-tryptophan modules, or other domainsthat allow purification on immobilized metals; maltose binding protein;protein A domains that allow purification on immobilized immunoglobulin;or the domain utilized in the FLAGS extension/affinity purificationsystem (Immunex Corp, Seattle Wash.).

In one embodiment, the fusion protein comprises a peptide or protein tag(e.g., for protein purification or detection). Peptide/protein tags areknown in the art, such as those described in Johnson, “Protein/PeptideTags,” DOI//dx.doi.org/10.13070/mm.en.2.116 including but not limited togreen fluorescent protein (GFP), FLAG, Myc epitope, polyhistidine,glutathione-S-transferase (GST), HA, V5, ABDz1-tag, Adenylate kinase(AK-tag), BC2-tag, Calmodulin-binding peptide, CusF, Fc, Fh8, Halo tag,Heparin binding peptide (HB-tag), Ketosteroid isomerase (KSI),maltose-binding protein (MBP), thioredoxin, PA (NZ-1), Poly-Arg,Poly-Lys, S-tag, SBP/Streptavidin-Binding Peptide, SNAP, Strep-II(Twin-Strep), and SUMO/SUMO2.

Affinity tags are a type of protein tag that is appended to proteins sothat they can be purified from their crude biological source using anaffinity technique. Affinity tags are known in the art, such as thosedescribed in Kimple et al. Curr Protoc Protein Sci.; 73: Unit-9.9.doi:10.1002/0471140864.ps0909s73. These include polyhistidine, GST, MBP,Calmodulin-binding peptide, intein-chitin binding domain,Streptavidin/Biotin-based tags, and His-Patch ThioFusion (thioredoxin).Affinity tags include small (e.g., 20 or less amino acid residues) orlarge affinity tags. Examples of small affinity tags include His, FLAG,Strep II, and S-peptide, and examples of large affinity tags includeMBP, GST, cellulose binding domains, calmodulin binding peptide, andHis-patch thioredoxin.

Affinity tags include epitope tags and reporter tags. Reporter tagsserve as reporters of protein expression and protein-proteininteraction. Reporter tags include, but are not limited to, enzymes suchas β-galactosidase (β-gal), alkaline phosphatase (AP), chloramphenicolacetyl transferase (CAT), and horseradish peroxidase (HRP).

Epitope tags include FLAG, hemagglutinin (HA), c-myc, T7, and Glu-Glu,which are used for the detection of fusion proteins in vitro and in cellculture. Their short, linear recognition motifs rarely affect theproperties of the protein of interest and are usually very specific fortheir respective primary antibodies. If the anti-myc antibody is used,specificity can be increased by using an enzyme-linked secondaryantibody to detect a conjugated anti-myc primary antibody instead ofusing an HRP- or AP-anti-myc conjugate alone.

Tags can be at either end of the target protein. Some epitope tags, suchas FLAG, are often used in tandem to increase their desired features, orin combination with another tag, such as in the construct of His-Myc andHis-V5.

Tandem affinity purification (TAP) is a dual-affinity purificationmethod based on the fusion of two affinity tags to a protein ofinterest, which allows purification of a tagged protein and isolation ofprotein complexes interacting with the protein of interest. The use ofTAP is encompassed within the present invention.

In one embodiment, the fusion protein comprises a histidine tag thatcomprises 2-10 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) histidine residues.For example, the histidine tag can comprise 6 histidine residues.

The inclusion of a cleavable linker sequences such as Factor Xa (see,e.g., Ottavi, Biochimie 80:289-293 (1998)), subtilisin proteaserecognition motif (see, e.g., Polyak, Protein Eng. 10:615-619 (1997));enterokinase (Invitrogen, San Diego, Calif.), and the like, between thetranslocation domain (for efficient plasma membrane expression) and therest of the newly translated polypeptide may be useful to facilitatepurification. For example, one construct can include a polypeptideencoding a nucleic acid sequence linked to six histidine residuesfollowed by a thioredoxin, an enterokinase cleavage site (see, e.g.,Williams, Biochemistry 34:1787-1797 (1995)), and an C-terminaltranslocation domain. The histidine residues facilitate detection andpurification while the enterokinase cleavage site provides a means forpurifying the desired protein(s) from the remainder of the fusionprotein. Technology pertaining to vectors encoding fusion proteins andapplication of fusion proteins are well described in the scientific andpatent literature, see, e.g., Kroll, DNA Cell. Biol. 12:441-53 (1993).

The fusion protein can contain one or more linkers (e.g., flexiblelinkers, rigid linkers, and in vivo cleavable linkers). Besides thebasic role in linking the functional domains together (as in flexibleand rigid linkers) or releasing free functional domain in vivo (as in invivo cleavable linkers), linkers offer many other advantages for theproduction of fusion proteins, such as improving biological activity,increasing expression yield, and achieving desirable pharmacokineticprofiles. Linkers are known in the art (see, e.g., Chen et al., Adv DrugDeliv Rev. 65(10): 1357-1369 (2013)).

Flexible linkers are used when the joined domains require a certaindegree of movement or interaction. They are generally composed of small,non-polar (e.g. Gly) or polar (e.g. Ser or Thr) amino acids. The smallsize of these amino acids provides flexibility, and allows for mobilityof the connecting functional domains. The incorporation of Ser or Thrcan maintain the stability of the linker in aqueous solutions by forminghydrogen bonds with the water molecules, and therefore reduces theunfavorable interaction between the linker and the protein moieties.

The most commonly used flexible linkers have sequences consistingprimarily of stretches of Gly and Ser residues (“GS” linker). An exampleof the most widely used flexible linker has the sequence of(Gly-Gly-Gly-Gly-Ser)_(n) (SEQ ID NO: 69). By adjusting the copy number“n”, the length of this GS linker can be optimized to achieveappropriate separation of the functional domains, or to maintainnecessary inter-domain interactions. Besides the GS linkers, many otherflexible linkers have been designed for recombinant fusion proteins.These flexible linkers are also rich in small or polar amino acids suchas Gly and Ser, but can contain additional amino acids such as Thr andAla to maintain flexibility, as well as polar amino acids such as Lysand Glu to improve solubility.

Rigid linkers keep a fixed distance between the domains and to maintaintheir independent functions. Examples of rigid linkers include alphahelix-forming linkers with the sequence of (EAAAK)_(n) (SEQ ID NO: 70)and linkers with a Pro-rich sequence, (XP)_(n), with X designating anyamino acid, preferably Ala, Lys, or Glu.

The polypeptide of the present invention also can contain a signalpeptide (i.e., a signal sequence, targeting signal, localization signal,localization sequence, transit peptide, leader sequence, or leaderpeptide), which is a short peptide present at the N-terminus oroccasionally C-terminus of most newly synthesized proteins that aredestined toward the secretory pathway. These proteins include those thatreside either inside certain organelles (the endoplasmic reticulum,Golgi or endosomes), secreted from the cell, or inserted into mostcellular membranes. Exemplary signal peptides are known in the art and aperson of ordinary sill in the art would recognize how to select aparticular signal peptide for use in the invention.

As used herein, “at least 80% identity” with reference to an amino acidsequence or a nucleotide sequence refers to 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%. 98%, or99% or more identity.

As used herein, examples of “an amino acid sequence modified bydeletion, insertion, substitution, or addition of one or more aminoacids” include an amino acid sequence modified by deletion, insertion,substitution, or addition of 1 or more to 30 or less, preferably 20 orless, more preferably 10 or less, and further preferably 5 or less aminoacids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or any rangesthereof). As used herein, examples of “a nucleotide sequence modified bydeletion, insertion, substitution, or addition of one or morenucleotides” include a nucleotide sequence modified by deletion,insertion, substitution, or addition of 1 or more to 90 or less,preferably 60 or less, more preferably 30 or less, further preferably 15or less, and further more preferably 10 or less nucleotides (e.g., 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, or any rangesthereof).

For example, in sequence comparison, typically one sequence acts as areference sequence, to which test sequences are compared. When using asequence comparison algorithm, test and reference sequences are enteredinto a computer, subsequence coordinates are designated, if necessary,and sequence algorithm program parameters are designated. Defaultprogram parameters can be used, as described below for the BLASTN andBLASTP programs, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters.

A “comparison window,” as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 20 to 600, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are well known in the art. Optimal alignment of sequencesfor comparison can be conducted, e.g., by the local homology algorithmof Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homologyalignment algorithm of Needleman & Wunsch, J Mol. Biol. 48:443 (1970),by the search for similarity method of Pearson & Lipman, Proc. Natl.Acad Sci. USA 85:2444 (1988), by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,Wis.), or by manual alignment and visual inspection (see, e.g., CurrentProtocols in Molecular Biology (Ausubel et al., eds. 1995 supplement)).

A preferred example of an algorithm that is suitable for determiningpercent sequence identity and sequence similarity are the BLAST andBLAST 2.0 algorithms, which are described in Altschul at al., Nuc. AcidsRes. 25:3389-3402 (1977) and Altschul et al., J Mol. Biol. 215:403-410(1990), respectively. Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Information.This algorithm involves first identifying high scoring sequence pairs(HSPs) by identifying short words of length W in the query sequence,which either match or satisfy some positive-valued threshold score Twhen aligned with a word of the same length in a database sequence. T isreferred to as the neighborhood word score threshold (Altschul et al.,Altschul et al., Nuc. Acids Res. 25:3389-3402 (1977) and Altschul etal., J Mol. Biol. 215:403-410 (1990)). These initial neighborhood wordhits act as seeds for initiating searches to find longer HSPs containingthem. The word hits are extended in both directions along each sequencefor as far as the cumulative alignment score can be increased.Cumulative scores are calculated using, for nucleotide sequences, theparameters M (reward score for a pair of matching residues; always >0)and N (penalty score for mismatching residues; always <0). For aminoacid sequences, a scoring matrix is used to calculate the cumulativescore. Extension of the word hits in each direction are halted when: thecumulative alignment score falls off by the quantity X from its maximumachieved value; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, T,and X determine the sensitivity and speed of the alignment. The BLASTNprogram (for nucleotide sequences) uses as defaults a wordlength (W) of11, an expectation (E) or 10, M=5, N=−4 and a comparison of bothstrands. For amino acid sequences, the BLASTP program uses as defaults awordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoringmatrix (see Henikoff & Henikoff, Proc. Natl. Acad Sci. USA 89:10915(1989)) alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and acomparison of both strands.

Another example of a useful algorithm is PILEUP. PILEUP creates amultiple sequence alignment from a group of related sequences usingprogressive, pairwise alignments to show relationship and percentsequence identity. It also plots a so-called “tree” or “dendogram”showing the clustering relationships used to create the alignment (see,e.g., FIG. 2). PILEUP uses a simplification of the progressive alignmentmethod of Feng & Doolittle, J Mol. Evol. 35:351-360 (1987). The methodused is similar to the method described by Higgins & Sharp, CABIOS5:151-153 (1989). The program can align up to 300 sequences, each of amaximum length of 5,000 nucleotides or amino acids. The multiplealignment procedure begins with the pairwise alignment of the two mostsimilar sequences, producing a cluster of two aligned sequences. Thiscluster is then aligned to the next most related sequence or cluster ofaligned sequences. Two clusters of sequences are aligned by a simpleextension of the pairwise alignment of two individual sequences. Thefinal alignment is achieved by a series of progressive, pairwisealignments. The program is run by designating specific sequences andtheir amino acid or nucleotide coordinates for regions of sequencecomparison and by designating the program parameters. Using PILEUP, areference sequence is compared to other test sequences to determine thepercent sequence identity relationship using the following parameters:default gap weight (3.00), default gap length weight (0.10), andweighted end gaps. PILEUP can be obtained from the GCG sequence analysissoftware package, e.g., version 7.0 (Devereaux et al., Nuc. Acids Res.12:387-395 (1984) encoded by the genes were derived by conceptualtranslation of the corresponding open reading frames.

The polynucleotides encoding the polypeptides of the present inventioncan be synthesized chemically or by genetic engineering based on theamino acid sequence of a Myd. For example, the polynucleotide can besynthesized chemically based on the amino acid sequence of thepolypeptides of the present invention or preprotein thereof. A contractsynthesis service of nucleic acid (provided from, for example, Medical &Biological Laboratories Co., Ltd., Genscript etc.) can be used for thechemical synthesis of the polynucleotide. Further, the synthesizedpolynucleotide can be amplified by PCR and cloning etc.

The polypeptides of the present invention can be produced, for example,by expressing a gene encoding a Myd polypeptide of the presentinvention. Preferably, a Myd polypeptide of the present invention can beproduced from a transformant in which the polynucleotide encoding a Mydpolypeptide of the present invention is introduced. For example, a Mydpolypeptide of the present invention is produced from a polynucleotideencoding a Myd polypeptide of the present invention introduced in atransformant after a polynucleotide encoding a Myd polypeptide of thepresent invention or a vector comprising it is introduced into a host toobtain a transformant and the transformant is cultured in an appropriatemedium. The proteins of the present invention can be obtained byisolating or purifying the produced Myd polypeptide from the culture.

Therefore, the present invention further provides a polynucleotideencoding a Myd polypeptide of the present invention and a vectorcomprising it. The present invention further provides a method ofmanufacturing a transformant, comprising introducing a polynucleotideencoding a Myd polypeptide of the present invention or a vectorcomprising it into a host. The present invention further provides atransformant comprising a polynucleotide encoding a Myd polypeptide ofthe present invention or a vector comprising it introduced from theoutside of a cell. The present invention further provides a method ofmanufacturing a Myd polypeptide of the present invention, comprisingculturing the transformant.

The present invention also includes the polynucleotides of theinvention, operably linked to a heterologous regulatory element. Theinvention may include an expression cassette or vector comprising thepolynucleotides of the present invention, and a host cell transformedwith a vector of the invention.

Alternatively, the polynucleotide encoding a Myd polypeptide of thepresent invention can be produced by introducing a mutation into thepolynucleotide synthesized according to the procedure with knownmutagenesis methods such as the ultraviolet irradiation andsite-directed mutagenesis. For example, the polynucleotide encoding thepolypeptides of the present invention can be obtained by introducing amutation into the polynucleotide of SEQ ID NO:1 or SEQ ID NO:2 with aknown method, expressing the obtained polynucleotide, investigating theexpressed protein's sweet-modification activity, and selecting apolynucleotide encoding the protein having desired sweet modificationactivity.

Site-directed mutagenesis of a polynucleotide can be performed with anymethods such as, for example, inverse PCR and annealing (Muramatsu etal. edit., “Revised 4th edition New genetic engineering handbook”,YODOSHA, p. 82-88). A variety of commercially available kits forsite-directed mutagenesis such as QuickChange II Site-DirectedMutagenesis Kit from Stratagene and QuickChange Multi Site-DirectedMutagenesis Kit can be used as needed.

Examples of the type of a vector comprising the polynucleotide encodingthe polypeptides of the present invention include, without limitation, avector usually used for gene cloning, for example, a plasmid, a cosmid,a phage, a virus, a YAC and a BAC. Examples of vectors include plasmids(e.g., DNA plasmids), yeast (e.g., Saccharomyces), and viral vectors,such as poxvirus, retrovirus, adenovirus, adeno-associated virus, herpesvirus, polio virus, alphavirus, baculovirus, Sindbis virus, plantviruses (e.g., Alphaflexiviridae or Potyviridae), and insect viruses(e.g., Baculoviridae).

Among these, a plasmid vector is preferred and for example acommercially available plasmid vector for protein expression, forexample, pUC19, pUC118, pUC119, pBR322 etc. (all of which are fromTAKARA BIO INC.) can be used.

The vector can comprise a DNA region comprising a replication initiationregion or a replication origin of DNA. Alternatively, a regulatorysequence such as a promoter region for initiating transcription of thegene, a terminator region or a secretory signal region for secreting anexpressed protein to the outside of a cell can be operably liked to theupstream of the polynucleotide encoding the proteins of the presentinvention (i.e. the MYD gene of the present invention) in the vector. Asused herein, a gene and a regulatory sequence being “operably liked”refers to a condition in which the gene and the regulatory region arepositioned so that the gene can be expressed under the regulation by theregulatory region.

The type of the regulatory sequence of a promoter region, a terminator,and a secretory signal region etc. is not specifically limited, and apromoter and a secretory signal sequence usually used can be selected touse as appropriate depending on the host into which the sequence isintroduced. For example, preferred examples of the regulatory sequencewhich can be incorporated to the vector of the present invention includethe cbh1 promoter sequence derived from Trichoderma reesei (Curr, Genet,1995, 28 (1): 71-79).

Alternatively, a marker gene to select a host into which the vector isappropriately introduced (for example, a resistance gene to an agentsuch as ampicillin, neomycin, kanamycin and chloramphenicol) can befurther incorporated into the vector of the present invention.Alternatively, a gene encoding a synthase of a required nutrient can beincorporated into the vector as a marker gene, when an auxotrophicstrain is used as a host. Alternatively, a related gene of themetabolism can be incorporated into the vector as a marker gene, when aselective medium requiring specific metabolism for growth is used.Examples of such a metabolism related gene include an acetamidase genefor using acetamide as a nitrogen source.

Ligation between the polynucleotide encoding a Myd polypeptide of thepresent invention and a regulatory sequence and a marker gene can beperformed by a known method in the art such as SOE (splicing by overlapextension)-PCR (Gene, 1989, 77: 61-68). The procedure for introducing aligated fragment into a vector is known in the art.

Examples of a host of a transformant into which the vector is introducedinclude a microorganism such as a bacterium and filamentous fungus.Examples of the bacterium include Escherichia coli and a bacteriumbelonging to Staphylococcus, Enterococcus, Listeria and Bacillus, ofwhich Escherichia coli and Bacillus bacteria (for example, Bacillussubtilis or a mutant thereof) are preferred. Examples of the Bacillussubtilis mutant can include protease 9 double deficient strain KA8AXdescribed in J. Biosci. Bioeng., 2007, 104 (2): 135-143 and a DBPAstrain, a mutant from protease 8 double deficient strain described inBiotechnol. Lett., 2011, 33 (9): 1847-1852, of which protein foldingefficiency is improved. Examples of the filamentous fungus includeTrichoderma, Aspergillus and Rhizopus. Also, for example, Pichiapastoris, Saccharomyces cerevisiae, Hansenula polymorpha, Yarrowialipolytica, Schizosaccharomyces pombe, Kluyveromyces lactis areappropriate expression hosts. In yet another aspect, the inventionincludes a host cell comprising one or more of the expression cassettesdescribed herein operably linked to control elements compatible withexpression in the cell. The cell can be, for example, a mammalian cell(e.g., BHK, VERO, HT1080, 293, RD, COS-7, or CHO cells), an insect cell(e.g., Trichoplusia ni (Tn5) or Sf9), a bacterial cell, a plant cell, ora yeast cell.

The recombinantly expressed polypeptides from Myd-encoding expressioncassettes are typically isolated from lysed cells or culture media.Purification can be carried out by methods known in the art includingsalt fractionation, ion exchange chromatography, gel filtration,size-exclusion chromatography, size-fractionation, and affinitychromatography. Immunoaffinity chromatography can be employed usingantibodies generated based on, for example, Gag antigens.

The invention provides a method of purifying a polypeptide havingsweet-taste modulation activity comprising (a) obtaining a compositioncomprising the polypeptide, and (b) purifying the composition viahydrophobic interaction chromatography (HIC) followed by size exclusionchromatography (SEC). In one aspect, the polypeptide comprises an aminoacid sequence having at least 80% sequence identity to a polypeptideselected from the group consisting of SEQ ID NO:3, SEQ ID NO:7, SEQ IDNO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ IDNO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17. Inanother aspect, the polypeptide has a polypeptide sequence having atleast 80% sequence identity to a polypeptide sequence selected from thegroup consisting of SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10,SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15,SEQ ID NO:16, and SEQ ID NO:17; (a) wherein the polypeptide contains atleast one substitution modification relative to the polypeptide sequenceselected from the group consisting of SEQ ID NO:3, SEQ ID NO:8, SEQ IDNO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ IDNO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17, wherein thepolypeptide is not the polypeptide of SEQ ID NO:3, or (b) wherein thepolypeptide further comprises a histidine tag, wherein the polypeptidehas sweet-taste modulation activity.

One of skill in the art is familiar with the purification techniques ofhydrophobic interaction chromatography (HIC) and size exclusionchromatography (SEC), including the selection of appropriate columns,buffers, and eluting solutions. Exemplary HIC and SEC purificationtechniques are described herein in Example 11. In an exemplary aspect,the purify of the polypeptide following purification by HIC and SEC isat least 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, orany ranges of values thereof.

The invention also contemplates a transgenic plant comprising aheterologous polynucleotide and/or heterologous polypeptide of theinvention as described herein. The plant has an altered phenotype due tothe expression of the heterologous nucleic acid sequence. The alteredphenotype may include a phenotype with increased sweetness in any plantpart, including fruits. The transgenic plant may contain an expressioncassette as defined herein as a part of the plant, the cassette havingbeen introduced by transformation of a plant with a vector of thisinvention. Such expression cassettes include regulatory sequences forexpression of heterologous coding sequences in plants, includingplant-expressible promoters and terminators. A transgenic plant can beany type of plant which can express the heterologous nucleic acidsequences described herein. The term “plant” includes whole plants,plant organs (e.g., leaves, stems, roots, etc.), seeds and plant cellsand progeny of same. The class of plants which can be used in the methodof the invention is generally as broad as the class of higher plantsamenable to transformation techniques, including both monocotyledonous(monocots) and dicotyledonous (dicots) plants. It includes plants of avariety of ploidy levels, including polyploid, diploid and haploid. Forexample, the transgenic plant can be an apple or strawberry.

Techniques for transforming a wide variety of plant species are wellknown in the art and described in the technical and scientificliterature. See, for example, Weising et al. (1988) Ann. Rev. Genet.,22:421-477 and Joung et al. (2015) “Plant Transformation Methods andApplications,” in Current Technology in Plant Molecular Breeding, (Kohet al., eds) Springer Dordrecht Heidelberg New York London, Chapter 9,pages 297-344. Any method known in the art for transformation of plantcells, including plant protoplasts, or plant tissue can be employed forplant transformation. Specific methods for plant transformation includeamong others, bolistic methods (gene guns), electroporation,microinjection, protoplast fusion and Agrobacterium-mediatedtransformation. Agrobacterium-mediated transformation can, for example,employ binary vectors that replicate in Escherichia coli andAgrobacterium tumefaciens or other Agrobacterium strains. A variety ofsuch binary vectors are known in the art and can be employed tointroduce heterologous polynucleotides into plant cells and planttissue. Plant expression vectors which include regulatory sequences forexpression of heterologous coding sequences, including plant-expressiblepromoter sequences and other plant regulatory sequences, in plant cellsand plant tissue are known in the art and can be employed to transformplants to express polypeptides as described herein.

A variety of plant-expressible promoters are known in the art and areavailable for use in heterologous constructs, vectors and transformedplant materials herein which contain polynucleotides encoding proteinhaving sweet-taste modulation activity. Plant-expressible promoters canderive from natural plant sources, plant virus sources and frombacteria, such as Agrobacterium strains, having promoters that areplant-expressible. Plant-expressible promoters include, among others,Cauliflower Mosaic Virus promoter (CaMV 35S), octopine and nopalinesynthase promoters (e.g., nos promoter), plant ubiquitin promoter (Ubi),rice actin promoter (Act-1), and maize alcohol dehydrogenase (Adh-1).Plant-expressible promoters include constitutive promoters, induciblepromoters, tissue-specific promoters, developmental stage-specificpromoters and examples of each type of promoter are known in the art.Tissue-specific promoters include, among others, those that directexpression in plant roots, plant leaves, fruit, flowers, pollen or cellsengaged in active photosynthesis (e.g., phosphoenolpyruvate promoters(PEP)). Development stage-specific promoters include those that directexpression during fruit ripening, flowering or seed set. Synthetic plantpromoters are also known in the art and are useful in heterologousconstructs, vectors and transformed plant materials (see, e.g., Ali S. &Kim W-C (2019) Frontiers in Plant Science, 10, article 1433).

Techniques for regeneration of plants from transformed protoplasts,plant cells, callus, or other plant tissue are well known in the art andcan be employed to regenerated whole plants and plant parts from suchtransformed plant material. Regeneration methods include organogenesisand embryogenesis. See: Handbook of plant cell culture. Volume 1:Techniques for propagation and breeding (1983) Edited by D. A. Evans etal., Macmillan (New York); R. H. Smith, Plant Tissue Culture: Techniquesand Experiments, 3^(rd) Edition (2012) Academic Press (New York); M. R.Davey & P. Anthony, Plant Cell Culture: Essential Methods (2010) JohnWiley & Sons (New York), particularly Chapters 3 and 9.

A method usually used in the field such as protoplast method andelectroporation can be used as a method of introducing a vector into ahost. A transformant of interest can be obtained by selecting a strainin which a vector is appropriately introduced using an index such as theexpression of a marker gene and/or auxotrophy.

Alternatively, a fragment in which the polynucleotide encoding a Mydpolypeptide of the present invention, a regulatory sequence and a markergene are ligated can be directly introduced into the genome of a host.For example, the polynucleotide encoding a Myd polypeptide of thepresent invention is introduced into the genome of a host byconstructing a DNA fragment added with a sequence complementary to thegenome of the host at both ends of the ligated fragment, introducing thefragment into the host and inducing homologous recombination between thehost genome and the DNA fragment by SOE-PCR.

Culturing the thus obtained transformant, in which the polynucleotideencoding a Myd polypeptide of the present invention or a vectorcomprising it is introduced, in an appropriate medium results in theexpression of the MYD cDNA on the vector, and then the production of aMyd polypeptide of the present invention. The medium used for theculture of such transformant can be selected depending on the type ofthe microorganism of such transformant by those skilled in the art asappropriate.

Alternatively, a Myd polypeptide of the present invention can beexpressed from the polynucleotide encoding a Myd polypeptide of thepresent invention or a transcription product thereof using a cell-freetranslation system. “Cell-free translation system” refers to an in vitrotranscription-translation system or an in vitro translation systemconstructed by adding reagents such as amino acids required for thetranslation of a protein into a suspension obtained by mechanicallydestructing cells to be a host.

A Myd polypeptide of the present invention produced in the culture orcell-free translation system can be isolated or purified by using ageneral method used for the purification of a protein, for example,centrifugation, ammonium sulfate precipitation, gel chromatography,ion-exchange chromatography and affinity chromatography etc. alone or incombination as appropriate. Here, when the gene encoding a Mydpolypeptide of the present invention and the secretory signal sequenceare operably liked on the vector within the transformant, the producedMyd polypeptide can be collected more easily from the culture becausethe Myd polypeptide is secreted to the outside of a cell. The Mydpolypeptide collected from the culture can be further purified withknown means.

The present invention also includes a method for producing a proteinhaving sweet-taste modulation activity, comprising culturing the hostcells of the invention in a medium under conditions that result inproducing the protein having sweet-taste modulation activity, similar toa known sweet flavoring agent or compound.

As used herein, a “sweet flavoring agent,” “sweet compound” or “sweetreceptor activating compound” refers to a composition that elicits adetectable sweet flavor in a subject, e.g., sucrose, fructose, glucose,and other known natural saccharide-based sweeteners, or known artificialsweeteners such as saccharine, cyclamate, aspartame, and the like as isfurther discussed herein, or a material that activates a T1R2/T1R3receptor in vitro. The subject may be a human or an animal.

A sweet flavoring agent or sweetening composition may be used in aneffective amount, which refers to an amount of a sweetening compositionof the invention that is sufficient to induce sweet taste in a subjectwhen present in a product for oral administration.

In an embodiment, the instant sweet proteins are capable of sweet-tastemodulation activity. Myd polypeptides of the invention may have e.g.,functional, physical and chemical effects at taste receptors, such assweet taste receptors. “Sweet-taste modulation activity” may refer toinhibitory, activating, e.g., agonist or antagonist properties of apolypeptide of the invention, identified using in vitro and in vivoassays for taste transduction. Proteins with inhibitory activity maybind to, partially or totally block stimulation, decrease, prevent,delay activation, inactivate, desensitize, or down regulate tastetransduction, e.g., antagonists. Activating polypeptides may bind to,stimulate, increase, open, activate, facilitate, enhance activation,sensitize, or up regulate taste transduction, e.g., agonists. Activatingpolypeptides are preferred.

Sweet taste modulation also refers to enhancing the taste, such as asweet taste, of a particular product for oral administration whenadministered as a combination.

Sweet-taste modulation activity may be detected by methods known in theart, e.g., in vitro methods, or in vivo by animal or human sensorytesting. While not wishing to be bound to any particular theory, Myd isinvolved in sweet taste activation e.g., is an agonist of taste 1receptor member 2 (T1R2) and/or taste 1 receptor member 3 (T1R3).However, Myd agonizes other taste receptors, such as bitter, umami, sourand salty. Such functional effects can be measured by any means known tothose skilled in the art, e.g., measurement of binding to tastereceptors T1Rs via changes in spectroscopic characteristics (e.g.,fluorescence, absorbance, refractive index), hydrodynamic (e.g., shape),chromatographic, or solubility properties, patch clamping,voltage-sensitive dyes, whole cell currents, radioisotope efflux,inducible markers, transcriptional activation of T1R genes;ligand-binding assays; voltage, membrane potential and conductancechanges; ion flux assays; changes in intracellular second messengerssuch as cAMP, cGMP, and inositol triphosphate (IP3); changes inintracellular calcium levels; neurotransmitter release, and the like.

Sensory testing (human or animal) may also be employed to determinewhether a Myd candidate polypeptide has sweet-taste modulation activity.Sensory evaluation is a scientific discipline that analyses and measureshuman responses to the composition of food and drink, e.g. appearance,touch, odor, texture, temperature and taste. Measurements using peopleas the instruments are sometimes necessary. Selection of an appropriatemethod to determine sweetening can be determined by one of skill in theart, and includes, e.g., discrimination tests or difference tests,designed to measure the likelihood that two products are perceptiblydifferent. Responses from the evaluators are tallied for correctness,and statistically analyzed to see if there are more correct than wouldbe expected due to chance alone.

Sensory evaluation is a scientific discipline that analyses and measureshuman responses to the composition of food and drink, e.g. appearance,touch, odor, texture, temperature and taste. Measurements using peopleas the instruments are sometimes necessary. The food industry had thefirst need to develop this measurement tool as the sensorycharacteristics of flavor and texture were obvious attributes thatcannot be measured easily by instruments. Selection of an appropriatemethod to determine the organoleptic qualities, e.g., sweetness ofproteins disclosed in the instant invention can be determined by one ofskill in the art, and includes, e.g., discrimination tests or differencetests, designed to measure the likelihood that two products areperceptibly different. For sweetness perception, for example, samplesof, for example, one or more of 5% sucrose, 6% sucrose, 7% sucrose, 8%sucrose, 9% sucrose, 10% sucrose, and a test sample can be ranked bytrained panelists in order of sweet taste intensity from low sweet tohigh sweet. In the instant invention, it should be understood that thereare any number of ways one of skill in the art could measure the sensorydifferences.

Brix measurement (or Brix scale) is a well-known application in the foodand beverage industry that determines pure sucrose content in water: 1degree Brix (° Bx)=1 g of sucrose/100 g of solution and represents thestrength of the solution as percentage by mass. 8° Bx is equivalent toapproximately an 8% sugar solution. As described in the Examples,purified polypeptide corresponding to SEQ ID NO:21 was tasted at 0.03mg/ml by a trained sensory scientist (0.2 mL aliquot) and found to havea sweetness equivalent to 8° Bx (approximately 8% sugar solution) (seeExamples 4, 5, 9, and 10).

In some embodiments, the Myd polypeptides of the invention includepolypeptides that are at least as sweet as sugar (on a w/w basis) (e.g.,1×), or alternatively, are 2×, 5×, 10×, 50×, 100×, 200×, 400×, 600×,800×, 1000×, 1500×, 2000×, 3000×, 5000×, 10,000×, 20,000× or moresweeter than sugar, as measured by any of the methods described above orknown in the art. In other embodiments, the Myd polypeptides are atleast 1% (at least 5%, at least 10%, at least 15%, at least 20%, atleast 25%, at least 30%, at least 35%, at least 40%, at least 45%, atleast 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, or at least 95%) assweet as sugar.

Foods, Beverages, Supplements, Medicinal Products

In one embodiment, the present invention includes a compositioncomprising, consisting essentially of, or consisting of a combination ofa product for oral administration and a sweetening compositioncomprising an isolated Myd polypeptide according to the invention, asdescribed herein. In one aspect, the combination has enhanced sweettaste compared to a product for oral administration lacking the Mydpolypeptide (control). In one embodiment, the product for oraladministration is not Mattirolomyces terfezioides truffle. The term“consisting essentially of” allows for the inclusion of components thatare not essential to the function or activity of the product and do notmaterially affect the function or activity, such an anti-caking agent,filler, stabilizer (e.g., thermal stabilizer), and bulking agent (e.g.,maltodextrose, gum acacia and the like).

In one embodiment, compositions comprising an isolated Myd protein ofthe invention may include a formulation that provides enhancedfunctionality to the isolated Myd protein. For example, a compositionmay include a formulation that stabilizes the Myd protein againstthermal, osmotic, pH, or other types of degradation. In one embodiment,the formulation stabilizes the Myd protein against thermal degradation.Exemplary compounds for stabilizing the Myd protein includes, forexample, L-arginine glycine, L-proline, L-histidine, β-alanine,L-serine, L-arginine ethyl ester dihydrochloride, L-argininamidedihydrochloride, 6-aminohexanoic acid, gly-gly, gly-gly-gly, tryptone,betaine monohydrate, D-(+)-trehalose dihydrate, xylitol, D-sorbitol,sucrose, hydroxyectoine, trimethylamine n-oxide dihydrate,methyl-α-d-glucopyranoside, triethylene glycol, sperminetetrahydrochloride, spermidine, 5-aminovaleric acid, glutaric acid,adipic acid, ethylenediamine dihydrochloride, guanidine hydrochloride,urea, N-methylurea, N-ethylurea, N-methylformamide, hypotaurine, TCEPhydrochloride, GSH (1-glutathione reduced), benzamidine hydrochloride,ethylenediaminetetraacetic acid disodium salt dihydrate, magnesiumchloride hexahydrate, cadmium chloride hydrate, non detergentsulfobetaine 195 (ndsb-195), non detergent sulfobetaine 201 (NDSB-201),non detergent sulfobetaine 211 (NDSB-211), non detergent sulfobetaine221 (NDSB-221), non detergent sulfobetaine 256 (NDSB-256), taurine,acetamide, oxalic acid dihydrate, sodium malonate pH 7.0, succinic acidpH 7.0, tacsimate pH 7.0, tetraethylammonium bromide, choline acetate,1-ethyl-3-methylimidazolium acetate, 1-butyl-3-methylimidazoliumchloride, ethylammonium nitrate, ammonium sulfate, ammonium chloride,magnesium sulfate hydrate, potassium thiocyanate, gadolinium(III)chloride hexahydrate, cesium chloride, 4-aminobutyric acid (GABA),lithium nitrate, DL-malic acid pH 7.0, lithium citrate tribasictetrahydrate, ammonium acetate, sodium benzenesulfonate, sodiump-toluenesulfonate, sodium chloride, potassium chloride, sodiumphosphate monobasic monohydrate, sodium sulfate decahydrate, lithiumchloride, sodium bromide, glycerol, ethylene glycol, polyethylene glycol200, polyethylene glycol monomethyl ether 550, polyethylene glycolmonomethyl ether 750, formamide, polyethylene glycol 400,pentaerythritol ethoxylate (15/4 EO/OH), 1,2-propanediol, polyethyleneglycol monomethyl ether 1,900, polyethylene glycol 3,350, polyethyleneglycol 8,000, polyvinylpyrrolidone k15, polyethylene glycol 20,000,(2-hydroxypropyl)-β-cyclodextrin, α-cyclodextrin, β-cyclodextrin,methyl-β-cyclodextrin.

In one aspect, the Myd polypeptide of the sweeting composition cancomprise an amino acid sequence having at least 80% sequence identity toSEQ ID NO:3 (or, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10,SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15,SEQ ID NO:16, or SEQ ID NO:17). In another aspect, the Myd polypeptidehas a polypeptide sequence having at least 80% sequence identity to apolypeptide sequence selected from the group consisting of SEQ ID NO:3,SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO: 11, SEQ ID NO:12, SEQID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17;(a) wherein the polypeptide contains at least one substitutionmodification relative to the polypeptide sequence selected from thegroup consisting of SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10,SEQ ID NO: 11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15,SEQ ID NO:16, and SEQ ID NO:17, wherein the polypeptide is not thepolypeptide of SEQ ID NO:3, or (b) wherein the polypeptide furthercomprises a histidine tag, wherein the polypeptide has sweet-tastemodulation activity. For example, the polypeptide comprises the aminoacid sequence of SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74,or SEQ ID NO:75 and does not consist of SEQ ID NO:3. In a particularaspect, the polypeptide comprises the amino acid sequence of SEQ ID NO:24, SEQ ID NO: 26, SEQ ID NO: 30, SEQ ID NO: 38, SEQ ID NO: 42, SEQ IDNO: 44, SEQ ID NO: 46, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO; 66,or SEQ ID NO: 68.

The present invention also includes a method for modulating the taste ofa product for oral administration, comprising combining the product fororal administration with an effective amount of an isolated Mydpolypeptide, as described herein. In one aspect, the combination hasenhanced sweet taste compared to a product for oral administrationlacking the Myd polypeptide (control). In one embodiment, the productfor oral administration is not Mattirolomyces terfezioides truffle.

In one aspect, the Myd polypeptide of the sweeting composition cancomprise an amino acid sequence having at least 80% sequence identity toSEQ ID NO:3 (or, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10,SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15,SEQ ID NO:16, or SEQ ID NO:17). In another aspect, the Myd polypeptidehas a polypeptide sequence having at least 80% sequence identity to apolypeptide sequence selected from the group consisting of SEQ ID NO:3,SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO: 11, SEQ ID NO:12, SEQID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17;(a) wherein the polypeptide contains at least one substitutionmodification relative to the polypeptide sequence selected from thegroup consisting of SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10,SEQ ID NO: 11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15,SEQ ID NO:16, and SEQ ID NO:17, wherein the polypeptide is not thepolypeptide of SEQ ID NO:3, or (b) wherein the polypeptide furthercomprises a histidine tag, wherein the polypeptide has sweet-tastemodulation activity.

The product for oral administration may be a food, a beverage, a dietarysupplement composition, or a pharmaceutical composition.

The term “product for oral administration” may refer to a comestibleproduct such as a food product, a beverage product; a medicinal(pharmaceutical) product, or a dietary supplement product such as anherbal supplement. As used herein, the term “medicinal product” includesboth solids and liquid compositions which are ingestible non-toxicmaterials which have medicinal value or comprise medicinally activeagents such as cough syrups, cough drops, aspirin and chewable medicinaltablets. An oral hygiene product is also a product for oraladministration and includes solids and liquids such as toothpaste ormouthwash.

In general terms, the present invention contemplates that food orbeverage products may include an isolated sweet protein of the inventionin an effective amount, e.g., in an amount of up to about 99% by weightrelative to the total weight of the food or beverage product, forexample in an amount from about 0.01% by weight to about 99% by weight.All intermediate weights (i.e., 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%,4%, . . . 90%, 95%, 99%) by weight relative to the total weight of thefood or beverage products are contemplated, as are all intermediateranges based on these amounts.

The compositions of the invention may include a “comestibly,biologically or medicinally acceptable carrier or excipient” which caninclude a solid or liquid medium and/or composition that is used toprepare a desired dosage form of a Myd polypeptide, in order toadminister a Myd polypeptide in a dispersed/diluted form, so that thebiological effectiveness of a Myd polypeptide is maximized. Acomestibly, biologically or medicinally acceptable carrier includes manycommon food ingredients, such as water at neutral, acidic, or basic pH,fruit or vegetable juices, vinegar, marinades, beer, wine, naturalwater/fat emulsions such as milk or condensed milk, edible oils andshortenings, fatty acids, low molecular weight oligomers of propyleneglycol, glyceryl esters of fatty acids, and dispersions or emulsions ofsuch hydrophobic substances in aqueous media, salts such as sodiumchloride, wheat flours, solvents such as ethanol, solid edible diluentssuch as vegetable powders or flours, or other liquid vehicles,dispersion or suspension aids, surface active agents, isotonic agents;thickening or emulsifying agents, preservatives, solid binders,lubricants and the like.

Food or beverage products that may be contemplated in the context of thepresent invention include baked goods; sweet bakery products,(including, but not limited to, rolls, cakes, pies, pastries, andcookies); pre-made sweet bakery mixes for preparing sweet bakeryproducts; pie fillings and other sweet fillings (including, but notlimited to, fruit pie fillings and nut pie fillings such as pecan piefilling, as well as fillings for cookies, cakes, pastries, confectionaryproducts and the like, such as fat-based cream fillings); desserts,gelatins and puddings; frozen desserts (including, but not limited to,frozen dairy desserts such as ice cream—including regular ice cream,soft serve ice cream and all other types of ice cream and frozennon-dairy desserts such as non-dairy ice cream, sorbet and the like);carbonated beverages (including, but not limited to, soft carbonatedbeverages); non-carbonated beverages (including, but not limited to,soft non-carbonated beverages such as flavored waters and sweet tea orcoffee based beverages); beverage concentrates (including, but notlimited to, liquid concentrates and syrups as well as non-liquidconcentrates, such as freeze-dried and/or powder preparations); yogurts(including, but not limited to, full fat, reduced fat and fat-free dairyyogurts, as well non-dairy and lactose-free yogurts and frozenequivalents of all of these); snack bars (including, but not limited to,cereal, nut, seed and/or fruit bars); bread products (including, but notlimited to, leavened and unleavened breads, yeasted and un-yeastedbreads such as soda breads, breads comprising any type of wheat flour,breads comprising any type of non-wheat flour (such as potato, rice andrye flours), gluten-free breads); pre-made bread mixes for preparingbread products; sauces, syrups and dressings; sweet spreads (including,but not limited to, jellies, jams, butters, nut spreads and otherspreadable preserves, conserves and the like); confectionary products(including, but not limited to, jelly candies, soft candies, hardcandies, chocolates and gums); sweetened breakfast cereals (including,but not limited to, extruded (kix type) breakfast cereals, flakedbreakfast cereals and puffed breakfast cereals); and cereal coatingcompositions for use in preparing sweetened breakfast cereals. Othertypes of food and beverage product not mentioned here but whichconventionally include one or more nutritive sweetener may also becontemplated in the context of the present invention.

As a consequence of the complete or partial replacement of nutritivesweeteners in the food or beverage products of the present invention,the food or beverage products of the present invention may be useful aslow calorie or dietetic products, medical foods/products (includingpills and tablets), and sports nutrition products, and may beparticularly suitable for food or beverage products requiring a lowersweetness at a given soluble solids level.

In some embodiments, the sweetening composition of the invention can besupplemented with other nutritional or non-nutritional sweeteners toform a sweetener system. The sweetener system may comprise thesweetening composition of the invention, a bulking agent such asmaltodextrose, gum acacia and the like, and at least one high intensitysweetener. The composition may be provided as liquid composition or adried blend.

In an embodiment, the present invention includes a process for enhancingthe sweet taste of a product for oral administration, comprising theaddition of a Myd polypeptide of the invention.

In another embodiment, the methods of the invention include a method forimproving the sweet flavor of a product for oral administration,comprising adding to the product for oral administration a sweeteningcomposition made by the methods of the invention. Amounts to add can bedetermined by methods known in the art, e.g., using sensory testing as aguide.

The following examples further illustrate the invention but, of course,should not be construed as in any way limiting its scope.

EXAMPLES Example 1

Fresh Mattirolomyces terfezioides truffles were obtained in situ usingappropriate procedures and permissions in their natural range. Freshsamples (29 in total) were shipped to MycoTechnology, Inc. facilitiesand were gently washed in RO water, then frozen in liquid nitrogen andstored at −80° C. The average moisture content of the truffles was 83.6%plus or minus 4.6%.

Aqueous extraction of the truffles was performed as follows. Eightdifferent samples of truffle were pestled in liquid nitrogen to grindinto a powder, then 5:1 v/w truffle of 4° C. water was added and allowedto incubate at 30 minutes at 4° C. The extracted material was thensubjected to low-speed brief centrifugation and the filtrate was tasted“neat.” The sweetness intensity was rated between 0 for no sweetness and10 for extremely sweet. Of the samples, the sweetness was rated asfollows:

TABLE 1 Sweetness of various truffle samples. Sweetness sample intensityNotes 1 5 Sweet taste at end 2 8 Sweet taste is upfront and intensifiesat mid-end and lingers 3 7 Sweetness is upfront and intensifies atmid-end and lingers. 4 5 Sweet upfront, low sweet linger 5 4 Sweetnessless strong 6 3 Low sweetness 7 6 A mild and clean sweet taste

Aqueous extract was stored at 4° C. at pH 7 and pH 2 in sodium phosphatebuffer, and little to no change in sweetness was observed over an 8-dayperiod.

Example 2

Purification of sweet protein Myd from M. terfezoides. FreshMattirolomyces terfezioides truffles were obtained in situ usingappropriate procedures and permissions in their natural range and storedat −80° C. 16.3 g sample was removed from the freezer, and was groundwith a mortar and pestle (white ceramic) in liquid nitrogen. Grindingproceeded for 15 minutes to fully pulverize tissue and obtain a finefrozen powder. Powder was added to 50 mL Falcon tubes and 20 mL RO—H₂Owas added, and vortexed to mix tissue, until no ice crystals wereobserved. A Rotor-Stator at a setting of 20 for 2×1 minutes at 4° C. wasused to breakup fragment and make a homogenous solution (H1). The volumeof slurry was adjusted to 53 mL with RO—H₂O and centrifuged at 7500×Gfor 30 minutes at 4° C. The supernatant (S1) from this step wascollected into 2 mL Eppendorf tubes and centrifuged in a 5417 R andEppendorf centrifuge at 20,000×G for 15 minutes at 4° C. The pellet (P1)was discarded. The sweet taste (via human sensory) was perceived in thesupernatant. Supernatant from this step was collected and pooled. Thesupernatant was then filtered through a 0.45 micron syringe filter(Cellulose, VWR International), 25 mm, and called S1+0.22 um Filtration.The filtrate was then washed with hexane (38 mL to 50 mL hexane 2 times,and the water phase was collected. S1FH is S1F+hexane wash. The hexanephase was saved and dried. The water phase was then precipitated withacetone (50 mL at −20° C. was added to 33 ml of fraction S1FH and setfor 30 minutes at −20° C.). The sample was centrifuged at 3,000×g andthe precipitate was collected. The precipitate was resuspended in 10 mMsodium phosphate pH 6, with the supernatant from this step called S2,and the precipitate called P2. The supernatant S2 was first applied toan AMICON centrifugal filter units with a molecular weight cutoff of 100kD, obtaining a filtrate (flow-through) portion (called 100F) and aretentate (called 100R); followed by applying the 100F to a unit with amolecular weight cutoff of 30 kD, resulting in a filtrate (30F) and aretentate (30R). The sweet taste fraction flowed through the 100 kDcolumn and appeared in 100F, and was retained on the 30 kD column (30R).A band was observed at approximately 13 kDa on the SDS-PAGE of FIG. 2,shown by the arrow, and this band was cut out and subjected toN-terminal sequence analysis by Edman degradation using standard methodsof detection e.g. liquid chromatography and mass spectrometry toidentify the residues for each cycle. A polypeptide was detected, SEQ IDNO:4.

Example 3 (Identification of RNA)

Sample Collection

Fresh Mattirolomyces terfezioides truffles were obtained in situ usingappropriate procedures and permissions in their natural range. A wildisolate (BDP2_18) of Mattirolomyces terfezioides truffle (gleba) wassourced from a natural environment. BDP2_18 was the largest wild trufflescavenged and the truffle had a sweetness characteristic of “sweetupfront, more fungal and earthy, low sweet linger.”

Sample Identification

The wild isolates of gleba were shipped frozen to GeneWiz for InternalTranscribed Spacer (ITS) Sequencing. The genome loci sequenced are theITS 1 & 2 region. The resulting sanger sequencing reads were thenaligned and trimmed of low-quality bases. Each sequence was thensubjected to an individual Basic Local Alignment Search Tool (BLAST)(Altschul, Gish, Miller, Myers, & Lipman, 1990) search to verifyidentity. BLASTn search was employed using nucleotide collection (nr/nt)with unpublished samples sequences excluded. The entry with the highestpercent identity to the wild isolate is Mattirolomyces terfezioidesstrain rib02.

Sample Preparation

The wild isolate, BPD2_18, was superficially washed with sterile waterand cut into cubes weighing ˜100 mg and flash frozen in liquid N2 to bestored at −80° C. Shipment was carried out on dry ice for GeneWiz.

RNA Sequencing

The following samples were submitted through GeneWiz for Standard RNASeqgoing through Illumina HiSeq, 2×150 bp, single index, per lane with˜350M raw paired-end reads per lane. This RNASeq study involved polyA+selection for transcriptome profiling.

GeneWiz extracted RNA using the Qiagen RNeasy Plus Universal mini kitfollowing manufacturer's instructions. RNA Library prep was done usingthe NEBNext Ultra RNA Library Prep Kit for Illumina. The Illuminaadapter sequences are outlined below.

(SEQ ID NO: 18) 5′-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3′ (SEQ ID NO: 19) 5′-GATCGGAAGAGCACACGTCTGAACTCCAGTCAC[i7barcode]ATCTCGTATGCCGTCTTCTGCTTG-3′

Fastq sequence files from the RNA-seq study on two strains ofMattirolomyces terfezioides (three replicates each) were trimmed andcleaned with HTStream to remove the following contaminants: PhiX (commoncontaminant from sequencing), rRNA reads, sequencing adapters, lowquality and “N” bases, poly-a tracts, primers and reads <50 bp.rnaSPAdes (Bushmanova E, Antipov D, Lapidus A, Prjibelski A D.rnaSPAdes: a de novo transcriptome assembler and its application toRNA-Seq data. Gigascience. 2019; 8(9):giz100.doi:10.1093/gigascience/giz100) was then used for the de novo assemblyof the transcriptome for each strain of M. terfezioides from the cleanedfiles. Bandage (a Bioinformatics Application for Navigating De novoAssembly Graphs Easily) (Ryan R. Wick, Mark B. Schultz, Justin Zobel,Kathryn E. Holt, Bandage: interactive visualization of de novo genomeassemblies, Bioinformatics, Volume 31, Issue 20, 15 Oct. 2015, Pages3350-3352) was used to visualize and search the assembly for the targetpeptide using a tBLASTn search (Gertz E M, Yu Y K, Agarwala R, SchafferA A, Altschul S F. Composition-based statistics and translatednucleotide searches: improving the TBLASTN module of BLAST. BMC Biol.2006; 4:41. Published 2006 Dec. 7. doi:10.1186/1741-7007-4-41) thatcompares the protein query sequence (PDLSSFITIKNNSNHVFTRT, SEQ ID NO:4)against the assembled transcriptome sequence database dynamicallytranslated in all six reading frames. Contigs that contained perfectmatches to the query protein sequence were analyzed for open readingframes (ORFs) and eventually used to construct the full-length mRNAconnected to the target peptide.

An RNA transcript was identified, and the DNA copy thereof has thesequence shown as SEQ ID NO:1, of which SEQ ID NO:2 is the predictedcoding sequence based on the start and stop codons. A predicted protein,SEQ ID NO:3, is also provided, having 121 amino acids with an estimatedsize of 13.3 kD. Length: 122 aa, molecular weight: 13.381 kDa, predictedisoelectric point: 8.64, and predicted charge at pH 7: 1.01.

Blast analyses. Identity between the predicted protein SEQ ID NO:3, andother protein sequences in GENBANK were 31% or less. A hypotheticalprotein from Pisolithus tincturius was found (GenBank: KIN98154.1; SEQID NO:7) with approximately 31% homology to SEQ ID NO:3; calledhypothetical protein M404DRAFT_1005519 [Pisolithus tinctorius Marx 270].It is hypothesized that SEQ ID NO:7 may also have sweet modulatingactivity. The complete cDNA copy of the RNA transcript is SEQ ID NO:5with the coding sequence given as SEQ ID NO:6.

Example 4 (Cloning and Heterologous Expression of Mycodulcein in E.coli; Confirmation of Sweet Taste.)

Based on the nucleotide sequence identified as SEQ ID NO:3, threeexpression vectors to express SEQ ID NO:20 containing a histidine tagwere synthesized and cloned by Atum, Inc. (Newark, Calif.) into threedifferent Atum vector backbones: pD454-SR (plasmid pMy_3000), pD454-MR(plasmid pMy_3001), and pD454-WR (plasmid pMy_3002), all of which are E.coli IPTG-inducible T7 promoter expression vectors with ampicillin-r,lacl, Lac01, Ori_pUC, and medium (M), strong (S) and weak (W) ribosomebinding sites on the plasmid. E. coli BL21 DE3 (Studier et al. (1986) J.Mol. Biol. 189:113-130) (New England Biolabs) was transformed withpMy_3000, pMy_3001, pMy_3002 using manufacturer protocols. In short,previously frozen competent cells were thawed and mixed with 1 pg-100 ngof plasmid DNA and held on ice for 30 minutes. A heat shock of 42° C.for 45 seconds was applied to this mixture. Immediately thereafter, themixture was placed into an ice bath lasting for 10 minutes. 950 μL ofpre-warmed LB media was added and then subjected to 1 hr of 225 rpmshaking at 37° C. Dilution of cells at 1:10 and 1:100 and plating 100 μLon the antibiotic plate was performed for each transformation reaction.An overnight growth at 37° C. allowed colonies to fully recover andbecome visible. To confirm successful transformation, csPCR (colonyscreen PCR) was performed to interrogate the cDNA region of the plasmidand expression was confirmed through lysate SDS-PAGE. Shake flask-scaleinduced expression was used to confirm heterologous expression in the E.coli host. This process yielded strains Z14CE, Z15CE, Z16CE containingthe plasmids pMy_3000, pMy_3001, and pMy_3002 respectively. The threestrains (Z14CE, Z15CE, Z16CE) were maintained on LB+ampicillin 100 μg/mLagar plates while the negative control (Eco_0001) was maintained on a LBagar plate. An overnight culture of was grown for each strain in 50 mLof LB liquid media in un-baffled 250 mL culture shake flask at 37° C.shaking 150 rpm overnight with appropriate antibiotics. Each overnightculture was subsequently seeded into 200 mL of TB liquid media thefollowing day into baffled 1000 mL culture shake flask at 37° C. shakingat 200 rpm and adjusted to 0.1 OD600 with the appropriate antibiotic.Once the OD600 reached 0.8, supplement of IPTG was added into the mediato a final concentration of 0.66 mM. Continued the shaking at 37° C. foran additional 5 hrs. After expression, the cells were centrifuged at4000 g for 20 min. The supernatant was discarded, and pellet wassuspended in ˜20 mL of wash buffer (10 mM Sodium Phosphate Buffer pH7.0). Suspended cells were disrupted in a high-pressure homogenizer (C3Emulsiflex, Avestin, Inc., Ottawa, ON, Canada) operated at a max of2,000 bar. Disrupted cells were centrifuged at 13,000 g (30 min), thesupernatant was collected, and the pellet was discarded. The supernatantcontaining solubilized protein was filtered through a 0.22 μm PESmembrane unit (Millipore, Burlington, Mass., USA). SDS PAGE was run anda 13.1 kD band (Coomassie stain) was observed to confirm expression.

Thermo Scientific™ HisPur™ Ni-NTA resin was used to purify thehis-tagged protein SEQ ID NO:20 using effective immobilized metalaffinity chromatography (IMAC). SEQ ID NO:20 was purified usingnickel-charged nitrilotriacetic acid (NTA) chelate immobilized onto 6%crosslinked agarose resin. Lysate was loaded onto prepared IMAC column,equilibrating to Binding Buffer: 20 mM sodium phosphate monobasic, 0.5Msodium chloride, 0.1 M imidazole, pH 7.4, and eluted using ElutionBuffer: 20 mM sodium phosphate monobasic, 0.5M sodium chloride, 0.5Mimidazole. The column was washed three times with Binding Bufferfollowed by eluting his-tagged mycodulcein four times with elutionbuffer, followed by ultrafiltration of eluted fractions using a 30 kDaMWCO filter followed by filtering filtrate through 10 kD filters for4000×G for 15 minutes. The retentate was diluted and spun again for awash step, repeated three times. Purification steps analysis by SDS-PAGEis shown in FIG. 3.

The purified fraction (shown in Lane 8 from FIG. 3, containing highlypurified SEQ ID NO:21) was tasted at 0.03 mg/ml by a trained sensoryscientist (0.2 mL aliquot) and found to have a sweetness equivalent to8° brix (approximately 8% sugar solution), confirming that mycodulceinisolated from M. terfezioides was responsible for the sweet tasteactivity observed in Examples 1 and 2. The sweet taste was verynoticeably sweet, with a “clean” sweetness (sugar-like taste) with noother flavors, with a slightly delayed onset and a sweet aftertaste.

Example 5 (Pilot-Scale Production of Mycodulcein (his-Tag))

E. coli strain Z14CE prepared in Example 4 containing coding sequenceSEQ ID NO:20 was tested for its performance during fermentation in alab-scale bioreactor. Bioreactor cultures were performed in 10.0 LBioflo 320 round bottomed stirred fermentor (BioFlo/CelliGen 310, NewBrunswick Scientific, Edison, N.J., USA). The fermenter was fitted withpH and dissolved oxygen sensors (Mettler Toledo, Ohio, USA). Temperaturewas controlled via a water-filled stainless-steel base. Agitation wasprovided by two mounted six-bladed Rushton turbines spaced 47 mm apartwith the lowermost impeller positioned just above the bottom of theshaft. Aeration occurred through a perforated pipe sparger ring.Dissolved oxygen (DO) was controlled at 20% of air saturation by using asequential cascade of agitation between 500 and 800 rpm and aerationbetween 5 and 8 L/min with air sparged at high-cell densities. The pHwas controlled at 7.0 using 5.0 M ammonium hydroxide. Antifoam 204(Sigma, St Louis, Mo., USA) was added automatically to control thefoaming. The latter was sensed using a conductivity probe mounted 10 cmabove the culture level. The main fermentation medium contained (perliter) 24 g yeast extract, 12 g tryptone, 5.42 mL glycerol, 100 mL ofphosphate buffer stock (0.17 M KH₂PO₄, 0.72 M K₂HPO₄) The medium wasadjusted to pH 7.0 using 2 M HCl. When the original glucose supply wasdepleted (signaled by a rise in pH), a feed medium (per liter)consisting of 200 g glucose, 21.1 g (NH₄)₂SO₄ and 19.7 g MgSO₄ waspumped into the fermenter at an initial flow rate of 1.00 mL/min. Unlessotherwise stated, the initial volume of the medium in the vessel was 4.0L. Inoculum (200 ml) consisted of a culture that had been grown for 16 hin a 1 L baffled shake flask (37° C., 200 rpm) in LB started culturemedia. The fermenter temperature was 37° C. Fermentations were inducedwith 0.66 mM IPTG after optical density reached 10-20 and continued forthereafter for 24 hours. The broth was subsequently centrifuged at 4000g for 20 min after the 24-hour induction period. The supernatant wasdiscarded, and pellet was suspended in 1 L of wash buffer (10 mM sodiumphosphate buffer pH 7.0). Suspended cells were disrupted in ahigh-pressure homogenizer (C3 Emulsiflex, Avestin, Inc., Ottawa, ON,Canada) operated up to a max of 1,500 bar. Disrupted cells werecentrifuged at 13,000 g (30 min), the supernatant was collected, and thepellet was discarded. The supernatant containing solubilized protein wasfiltered through a 0.22 μm PES membrane unit (Millipore, Burlington,Mass., USA).

The purified supernatant prepared in this Example was tasted at 0.03mg/ml by a trained sensory scientist (0.2 mL aliquot) and found to havea sweetness equivalent to 8° brix (approximately 8% sugar solution). Thesweet taste was very noticeably sweet, with a “clean” sweetness(sugar-like taste) with no other flavors, with a slightly delayed onsetand a sweet aftertaste.

Supernatant was stored in aliquots at −20° C. and used for furtherexperiments.

Example 6 (Mycodulcein from E. coli ELISA Quantification)

A direct ELISA was developed to quantify his-tagged mycodulcein (SEQ IDNO:21) with a horseradish peroxidase (HRP) conjugated antibody to the6×His-Tag sequence on the carboxy terminus of SEQ ID NO:21. The ELISAenables the measurement of mycodulcein concentration in complex lysatesand purified protein. Recombinant 6×His-tagged E. coli mycodulcein has amolecular weight of 14.2 kDa and a molar extinction coefficient of27,960 M⁻¹ cm⁻¹, calculated from the amino acid sequence by methodsknown in the art. Purity was assessed by SDS-PAGE as ≥98%. Mycodulceinprotein concentration was determined by absorbance at 280 nm usingBeer-Lambert's Law, then a standard curve was generated using knownconcentrations of mycodulcein (μg/ml) for the ELISA.

ELISA procedure: Protein was bound to the walls of a high-proteinbinding 96-well plate in 50 mM carbonate buffer pH 9.4 coating bufferfor 30 minutes at room temperature. The plates were washed 3 times withphosphate buffered saline (PBS) pH 7.4 with 0.02% Tween-20. Nonspecificbinding sites on the microplate were blocked with 5% BSA in PBS pH 7.4for 15 minutes at room temperature and washed three times with PBS 0.02%Tween-20. The primary antibody was diluted (1:1000) in blocking bufferand the microplates were incubated for 1 hour and washed 3 times in PBS0.02% Tween-20. The colorimetric substrate,3,3′,5,5′-tetramethylbenzidine (TMB), was used to develop the HRPreaction for 8 minutes and stopped with 2N sulfuric Acid.

Example 7 (Quantitative Characterization of the Concentration-Dependenceof Mycodulcein)

Opertech Bio (Philadelphia, Pa.) performed a quantitativecharacterization of the concentration-dependence of the taste propertiesof the purified his-tagged mycodulcein (SEQ ID NO:21) obtained from E.coli as described in Example 5 and purified as described in Example 4.The sweet-taste potency and relative efficacy of mycodulcein wascompared with sucrose and with other sweeteners thaumatin, rebaudiosideA, and aspartame. Control standards were solutions of 200 mM sucrose,100 mM NaCl, 0.5 mM quinine, and 10 mM citric acid. FIG. 4 shows theconcentration-response functions for sweet taste for mycodulcein,aspartame, thaumatin, rebaudioside A. Data are plotted as the proportion(p) of responses occurring on the 200 mM sucrose-associated (“sweet”)target. Each data point in the curves for mycodulcein, aspartame,thaumatin, rebaudioside A was calculated as the average across 32replicates and averaged across 16 replicates for the sucrose curve;error bars are SEM. Points for water and sucrose controls were similarlycalculated as the average across 128 and 64 replicates, respectively.Curves were fit by non-linear regression.

The concentrations that elicit half-maximal sweet taste response (EC50s,or potencies) were derived from the nonlinear regression. The EC50s (and95% confidence intervals) for mycodulcein (MYC), sucrose (SUC),aspartame (ASP), rebaudioside A (REB), and thaumatin (THN) are given inTable 2. Also provided are the equivalencies to sucrose on a molar basisand on a weight basis.

TABLE 2 Relative Relative EC50 to to (molar EC50 Sucrose Sucrose units)CI 95% (mg/ml) (mM) (mg/ml) MYC 2 μM 2-3 μM 0.0286 18000 431 THN 0.3 μM0.2-0.3 μM 0.0066 120000 1865 REB 42 μM 33-57 μM 0.0411 857 300 ASP 182μM 142-254 μM 0.0532 198 231 SUC 36 mM 27-45 mM 12.3120 1 1

Evaluation of thaumatin, sucrose, and SEQ ID NO:21 was carried out usinga CATA (click all that apply) time intensity of the sweet sensation ofnamed analytes in water at 0.045 mg/ml for proteins and 10% sucrose.Methodology: Time Intensity Technique; Data collection software:EyeQuestion, responses recorded every 2.57 seconds; Scaling Method: a15-point sweet scale, e.g. score 5=5% sucrose, score 10=10% sucrose;Evaluation Protocol: small volume sip, tilt and spit test. There were3-6 panelists and 2 replicates. All samples were blinded and presentedwith randomized 3-digit codes.

Training Strategies: An intense training on sweet scale over 6 weeks on15-point sweet scale to confidently assign sweet values. Due to uniquesweet behavioral patterns, it is necessary to train time intensityprinciples over 3 weeks. Samples were tasted with a stopwatch to notetimes and help reach a consensus. # of Panelists: 3-6, # ofreplications: 2. All samples were blinded and presented with randomized3-digit codes. Statistical analysis: due to the small # of panelists, astatistical analysis cannot be performed.

The maximum intensity (Imax) of mycodulcein and thaumatin showsapproximately 1 point higher on the 15 point scale at the amounts testedthan sucrose. Thaumatin and mycodulcein have a flatter slope, indicatinga longer peak time and more gradual/longer decline. Sucrose approachesthreshold sweetness (Intensity <1) at 162 seconds, sooner than thaumatinand mycodulcein. When sucrose reaches threshold level, thaumatin andmycodulcein are recognizable at a low-moderate intensity. Mycodulceinand thaumatin appeared to be of similar potency in this experiment,which is on the order of 3000× sweeter than sucrose on a weight basis,or approximately 120,000× sweeter than sucrose on a molar basis. Fromthese two experiments, mycodulcein is shown to be a high intensity sweetprotein with a potency of between 400× sweeter than sucrose and 3000×sweeter than sucrose on a weight basis.

Example 8 (Production and Testing of Variants of Mycodulcein forSweetness and Thermal Stability)

Method to identify potential key residues in mycodulcein. Although sweetproteins have no primary sequence identity, the overall tertiarystructures have a sweet finger motif (Tancredi, T., Pastore, A.,Salvadori, S., Esposito, V. & Temussi, P. A. Interaction of sweetproteins with their receptor: A conformational study of peptidescorresponding to loops of brazzein, monellin and thaumatin. EuropeanJournal of Biochemistry 271, (2004): 2231-2240.). Sweet proteins haveantiparallel beta-sheets with an alpha-helix perpendicular to thebeta-sheets. The sweet proteins thaumatin, monellin, brazzein, andlysozyme tertiary structures were analyzed using PyMOL 2.0 (The PyMOLMolecular Graphics System, Version 2.0 Schrödinger, LLC) and compared toa model of mycodulcein generated with Phyre2 (Kelley L A et al. ThePhyre2 web portal for protein modeling, prediction, and analysis NatureProtocols 10, (2015): 845-858) (FIG. 5A). Twenty-three conservativesingle point mutations of ionic amino acid residues were generated. Thenegatively charged glutamine and aspartic acids differ by an additionalcarbon in the aliphatic chain. Although substituting the positivelycharged lysine and arginine is considered a conservative substitution,the guanidinium of arginine can form additional interactions with aminoacids, including hydrogen bonds, aromatic, and aliphatic contacts. Theionic amino acid mutations were lysine to arginine, arginine to lysine,aspartic acid to glutamic acid, and glutamic acid to aspartic acid. Therelative positions of each mutant were modelled by PyMOL 2.0 and werecategorized as N-terminus, 3 loop regions, 5 beta-sheets, 1 alpha-helixand, the C-terminus (see FIG. 5B.)

Specifically, the following single mutants were generated, and theirpredicted location is in Table 3. See also FIG. 5B, which shows arepresentation of SEQ ID NO:3 predicted secondary structure superimposedon putative secondary structure motifs and location of the Table 3 pointmutations within each motif.

Cloning: Eco_0001 aka E. coli BL21 DE3 (E. coli str. B F⁻ ompT gal dcmIon hsdS_(B)(r_(B) ⁻m_(B) ⁻)λ(DE3 [lacI lacUV5-T7p07 ind1 sam7 nin5])[malB⁺]_(K-12)(λ^(S))) (obtained from New England Biolabs #C2527I), wastransformed with twenty-three plasmids (pMy_3018 to pMy 3040) usingmanufacturer protocols. In short, previously frozen competent cells werethawed and mixed with 1 pg-100 ng of plasmid DNA and held on ice for 30minutes. A heat shock of 42° C. for 45 seconds was applied to thismixture. Immediately thereafter, the mixture was placed into an ice bathlasting for 10 minutes. 950 μL of pre-warmed LB media was added and thensubjected to 1 hr of 225 rpm shaking at 37° C. Dilution of cells at 1:10and 1:100 and plating 100 μL on the antibiotic plate was performed foreach transformation reaction. An overnight growth at 37° C. allowedcolonies to fully recover and become visible. This process yieldedstrains Z18CE to Z40CE containing the plasmids pMy_3018 to pMy_3040sequentially. Shake flask-scale induced expression was used to confirmheterologous expression in the E. coli host. Post-transformationalmutants were plated and maintained on LB+Ampicillin 100 μg/mL agarplates.

Plates were incubated for 16 hours at 37° C. Colony screen PCR wasperformed to confirm genotypes using colony screening primers designedto interrogate the flanking region along with the cDNA region of theplasmid. A successful transformation resulted in a DNA fragment of acertain size while a negative control and no template control result inno PCR band. Successful transformation was observed for all mutants.

Shake flask-scale induced expression was used to confirm heterologousexpression in the E. coli host.

23 strains (Z38CE to Z60CE) were maintained on LB+ampicillin 100 μg/mLagar plates while the negative control (Eco_0001) was maintained on a LBagar plate. An overnight culture of was grown for each strain in 50 mLof LB liquid media in un-baffled 250 mL culture shake flask at 37° C.shaking 150 rpm overnight with appropriate antibiotics. Each overnightculture was subsequently seeded into 200 mL of TB liquid media thefollowing day into baffled 1000 mL culture shake flask at 37° C. shakingat 200 rpm and adjusted to 0.1 OD₆₀₀ with the appropriate antibiotic.Once the OD₆₀₀ reached 0.8, supplement of IPTG was added into the mediato a final concentration of 0.66 mM. Continued the shaking at 37° C. foran additional 5 hrs. Afterwards, cells were collected by centrifugationat 5000 g at 4° C. for 5 min. E. coli cells were washed once with colddH₂O and centrifuged again at 5000 g at 4° C. for 10 minutes. Forconfirmation of successful expression, cell lysate was created usingliquid nitrogen and a mortar and pestle. The cell pellet was resuspendedin 10 mL of cold dH₂O and the crude lysate was spun at 20,000 g at 4° C.for 5 minutes. Finally, the supernatant was aspirated and filteredthrough 0.2 μm PES filter and run on SDS-PAGE protein electrophoresis.Crude lysates were tasted in order to identify sweet-tasting mutants.Table 3 shows results of the testing.

TABLE 3 Results of sweetness testing on single point mutations ofmycodulcein PROTEIN SEQ AA Hypothesized Content Sweetness? ID NO:Substitution Location Control- Yes 21 N/A N/A no mutation Z38CE Yes 24D3E N-terminus, exterior surface Z39CE Yes 26 K11R Beta sheet 1 Z40CE No28 R20K Beta Sheet 1, exterior surface Z41CE Yes 30 K26R Linker regionbetween beta sheet 2 and beta sheet 3 Z42CE No 32 E35D Linker regionbetween beta sheet 2 and beta sheet 3 Z43CE No 34 K44R Beta sheet 3Z44CE No 36 D46E Beta sheet 3 Z45CE Yes 38 K51R Loop region 2 Z46CE No40 D52E Loop region 2 Z47CE Yes 42 R57K Loop region 2 Z48CE Yes 44 R66KBeta sheet 4 Z49CE Yes 46 D69E Loop region 3 Z50CE No 48 R75K Beta sheet5 Z51CE Yes 50 D85E Alpha helix Z52CE Yes 52 E86D Alpha helix Z53CE Yes54 E89D Alpha helix Z54CE No 56 D94E C-terminus Z55CE Yes 58 D97EC-terminus Z56CE Yes 60 K103R C-terminus Z57CE Yes 62 R106K C-terminusZ58CE Yes 64 R110K C-terminus Z59CE Yes 66 E117D C-terminus Z60CE Yes 68K120R C-terminus

16 of the variants (Z38CE, Z39CE, Z41CE, Z45CE, Z47CE, Z48CE, Z49CE,Z51CE, Z52CE, Z53CE, Z55CE, Z56CE, Z57CE, Z58CE, Z59CE, Z60CE) all ofwhich had sweet taste were additionally re-expressed using 200 mL ofmedia. After expression, the cells were centrifuged at 4000 g for 20 minafter the 24-hour induction period. The supernatant was discarded, andpellet was suspended in ˜20 mL of wash buffer (10 mM sodium phosphatebuffer pH 7.0). Suspended cells were disrupted in a high-pressurehomogenizer (C3 Emulsiflex, Avestin, Inc., Ottawa, ON, Canada) operatedat a max of 2,000 bar. Disrupted cells were centrifuged at 13,000 g (30min), the supernatant was collected, and the pellet was discarded. Thesupernatant containing solubilized protein was filtered through a 0.22μm PES membrane unit (Millipore, Burlington, Mass., USA). The materialwas subsequently isolated through IMAC Purification as described inExample 4.

The purified samples were tasted by a trained sensory scientist.Mycodulcein stocks were diluted to equal protein concentration asmeasured by ELISA. Subjects followed a sip and spit protocol approved byan Institutional Review Board. 0.2 ml of each purified mutant was placedon the tongue and intensity of sweetness perception, time of onset ofsweet perception, and duration of sweetness perception were noted.Results are shown in FIG. 6 and discussed hereinbelow.

Effects of Conservative Point Mutations on Mycodulcein Sweetness

To correlate the effects of conservative single point mutations onsweetness, published mutations of thaumatin, brazzein, monellin andlysozyme were compared to mycodulcein. Since the objective is to matchmycodulcein time and intensity profile to sucrose's, we measured sucroseequivalence, onset, and total duration by sensory analysis. Sucrose hasa fast onset, high intensity, and fast duration. Thus, mutations thatreduce onset and duration are desirable as are mutations that match orimprove sucrose equivalency. Mutations that increase onset and totalduration are undesirable, as are mutations that decrease sucroseequivalency. See FIGS. 5B and 6.

N-Terminus—Exterior—D3E

The conservative change of a single mutation of D3E at the exteriorN-terminus, resulted in the loss of sucrose equivalence and duration;however, the onset was only reduced by slightly. These results suggestthat the charge, size, and polarity of sweet proteins N-terminus isimportant for sweet taste and protein stability.

Beta-Sheet 1—Exterior—K11R

The conservative change of a single mutation at K11R, resulted in asmall increase in sucrose equivalence and a moderate reduction in onset;however, the duration of sweetness was dramatically increased. Theseresults suggest that K11 is an important residue for the binding to thesweet taste receptor T1R2/T1R3 and may affect the off rate ofmycodulcein from the receptor.

Region Between Beta Sheets 2 and 3—Exterior—K26R—Linker Region

The conservative mutation at K26 resulted in a slight decrease forsucrose equivalence, showing that this conservative mutation does nothave a significant effect on protein functionality.

Loop 2 Region—Exterior—K51R

Molecular modelling showed that all putative loop region mutations weresolvent exposed. Except for a moderated decrease in onset, only a smalldecrease in sucrose equivalence and total duration were observed insensory studies, showing that this conservative mutation does not have asignificant effect on protein functionality.

Loop 2 Region—Exterior—R57K

The mutation R57K has a significant inhibitory effect on onset, sucroseequivalence and total duration.

Beta Sheet 4—Exterior—R66K

Mutant R66K has a significant decrease on sucrose equivalence and totalduration, while increasing onset slightly. The R66K mutation may be akey residue for affinity and off rate on the sweet taste receptor.

Loop 3 Region—Exterior—D69E

A mutation at D69E has a small decrease for sucrose equivalency,duration, and small increase in duration. It is predicted that this areain this region is relatively insensitive to conservative mutations.

Alpha-Helix Region—Exterior—D85E and D86E

Both D85E and D86E have similar effects on mycodulcein organolepticproperties. Sucrose equivalence and duration for D85E and D86E werereduced. Onset was similar to the control.

C-terminus-exterior-D97E, K103R, R106K, and E117D had minimal effectscompared to the control suggesting that these areas are relativelyinsensitive to conservative mutations. However, R110K and K120R showeddecreased sweetness and duration, but onset was similar.

As shown in Table 3, the conservative single point mutations at R20K,E35D, K44R, D46E, D52E, R75K, and D94E, resulted in the loss of proteinexpression. These data suggest that these residues may be involved inprotein folding or expression within the E. coli host. The predictedtertiary structure model discussed above in this Example supportsprotein misfolding resulting from these changes, as all these residuesare contained in the predicted beta-sheets.

REFERENCES

-   Korz, D. J., Rinas, U., Hellmuth, K., Sanders, E. A. & Deckwer,    W.-D. Simple fed-batch technique for high cell density cultivation    of Escherichia coli. Journal of Biotechnology 39, 59-65 (1995).-   Norsyahida, A., Rahmah, N. & Ahmad, R. M. Y. Effects of feeding and    induction strategy on the production of BmR1 antigen in    recombinant E. coli. Letters in Applied Microbiology 49, 544-550    (2009).

Example 9

Thermal Stability was performed on protein samples from the IMACPurification of the 16 sweet mutants as described above in Example 8,normalized to 0.04 mg/mL, using the GloMelt™ Thermal Shift ProteinStability Kit (Biotium, Inc. Fremont, Calif.), using instructionsprovided by the manufacturer. In short, the individual reaction tomeasure thermal shift relies on mixing the following: mycodulcein, 36μg/ml in 25 mM sodium phosphate buffer pH 7.4 with reagents provided inthe kit per manufacturer's instructions. For the thermal shiftmeasurement, Bio-Rad CFX96 Touch system using BR Clear plates, scan-modeSYBER/FAM only, 25° C. for 30 seconds, melt curve 25° C. to 95° C.,increment 0.5° C. for 10 sec plus plate read was used. The Tm isdetermined based on the midpoint determined for a curve fitted to theexperimental data with a five-parameter equation using the techniques asdescribed in Schulz, M. N., Landström, J. & Hubbard, R. E. MTSA—A Matlabprogram to fit thermal shift data. Analytical Biochemistry 433, 43-47(2013). Results (Table 4) show that thermal stability is minimallyaffected by the amino acid changes in the mutants tested.

TABLE 4 Results of thermal stability testing for mutants PROTEIN SEQ IDNO: Mutant ID Tm 24 Z38CE 58.02135 26 Z39CE 58.9354 30 Z41CE 58.81657 38Z45CE 60.66312 42 Z47CE 58.48098 44 Z48CE 58.99634 46 Z49CE 59.79641 50Z51CE 59.99543 52 Z52CE 58.81274 54 Z53CE 57.96772 58 Z55CE 60.14034 60Z56CE 59.29316 62 Z57CE 61.10271 64 Z58CE 57.92335 68 Z60Ce 60.168

Example 10 (Cloning and Heterologous Expression of Mycodulcein inSaccharomyces cerevisiae; Confirmation of Sweet Taste)

Based on the nucleotide sequence identified as SEQ ID NO:3, twoexpression vectors to express SEQ TD NO:21 (pMy_4003) (his-tagged) andSEQ ID N0:3 (pMy_4002) (native) mycodulcein were synthesized and clonedby Atum, Inc. (Newark, Calif.) into Atum vector non-secretory backbonepD1234 containing URA3 marker, and strong constitutive promoter GPD. Thetransformation procedure involves making electrocompetent cells and thenintroducing expression vectors through electroporation. In short, theelectrocompetent cells are created by first growing the cells to betweenthe early and mid-log phase with multiple washes to remove the salt fromthe growth medium. After mixing 1-5 μg of the expression vector, thesample is subjected to the following settings on a Gene Pulser IIElectroporator (Charging Voltage: 1.5 kV, Capacitance: 25 μF,Resistance: 200Ω) 1 mL of prewarmed 30° C. YPD is added immediately, andthe suspension is incubated for 1-2 h at 30° C. shaking at 200-250 rpm.Post-transformational mutants were plated and maintained on SC-ura agarplates. This process yielded strains Z19ES, Z20ES containing theplasmids pMy_4002 and pMy_4003 respectively. csPCR (colony screen PCR)was performed to interrogate the cDNA region of the plasmid. Thus, asuccessful transformation would result in the DNA fragment of 303 bpwhile a negative control and no template control would result in no PCRband and expression was confirmed.

The two strains (Z19ES, Z20ES) were maintained on SC-ura agar plateswhile the negative control was maintained on a SC agar plates. Anovernight culture of was grown for each strain in 50 mL of SC-ura/SCliquid media in un-baffled 250 mL culture shake flask at 37° C. shaking150 rpm overnight. Each overnight culture was inoculated into 200 mL ofSC-ura/SC (O-RDL-R10_TB Media) liquid media in baffled 1000 mL cultureshake flask at 30° C. shaking at 200 rpm and adjusted to 0.02 OD600. Theshaking was continued at 30° C. for an additional 48 hrs. Afterwards,cells were collected by centrifugation at 5000 g at 4° C. for 5 min.Afterwards, S. cerevisiae cells were washed with cold dH2O andcentrifuged again at 5000 g at 4° C. for 5 minutes. For confirmation ofsuccessful expression, cells were lysed using liquid nitrogen and amortar and pestle. Cell pellets were resuspended in 10 mL of cold dH₂Oand lysate was spun at 20,000 g at 4° C. for 5 minutes. Supernatant wasfiltered with a 0.2 μm PES filter. The filtrate (both strains) wasconfirmed to taste sweet by methods described in Example 3.

Thermo Scientific™ HisPur™ Ni-NTA resin was used to purify thehis-tagged protein SEQ ID NO:20 from S. cerevisiae using effectiveimmobilized metal affinity chromatography (IMAC). SEQ ID NO:20 waspurified using nickel-charged nitrilotriacetic acid (NTA) chelateimmobilized onto 6% crosslinked agarose resin. Lysate was loaded ontoprepared IMAC column, the column was washed three times with 0.02 Mimidazole in PBS followed by eluting his-tagged mycodulcein four timeswith 0.3 M imidazole in PBS followed by ultrafiltration of elutedfractions using a 50 kDa MWCO filter followed by concentration anddesalting with a 3 kDa MWCO filter.

Example 11 (Purification of Native Mycodulcein from Strain Z19ES (S.cerevisiae))

Three chromatographical techniques were assessed for effectiveness forpurification of native mycodulcein (SEQ ID NO:3) expressed in S.cerevisiae: cation exchange (CIEX), hydrophobic interaction (HIC), andsize exclusion chromatography (SEC).

Cation Exchange Assessment.

The isoelectric point for the native mycodulcein was determined to be˜9.5 by isoelectric focusing, suggesting a cation exchange column mightbe successful in purifying mycodulcein. The clarified cell lysateprepared as described in Example 10 was mixed with 2× starting buffer toobtain a cell lysate in 50 mM sodium phosphate, 1M ammonium sulfate atpH 7.0, and it was stored at 4° C. for future use. The purificationprocedure was performed on ÄKTA Explorer 100 system (GE Healthcare,Sweden), and the eluted proteins were monitored at 280 nm and 215 nm ona UV detector UV-900 (GE Healthcare, Sweden). A PreDictor plate (GEHealthcare, Sweden) prefilled with CIEX resins was used to screenbinding conditions of native mycodulcein. The prefilled plate containsthree main resins: Capto S (strong CIEX), Capto MMC (Weak CIEX), and SPSepharose Fast Flow (strong CIEX). Lysate was dialyzed into 20 mM sodiumphosphate dibasic, and different pH values ranging from 4 to 9 werescreened and the optimal conditions were then scaled up using HiScreencolumn. Equilibration was conducted using 25 mM sodium phosphate dibasicat pH 5 at flow rate of 3 mL/min. Binding proteins were eluted by anincreasing sodium chloride gradient from 0 to 1 M using 25 mM sodiumphosphate dibasic, 1M sodium chloride at pH 7. Different binding andeluting conditions were screened in which the weak cation exchangerCapto MMC showed the best binding capabilities at pH 5. However, due tolow purity (25%) after this step, an alternative purification step wassought. FIG. 7A shows PAGE analysis of the eluted fraction from CaptoMMC. M: protein marker; lane 1: eluted fraction, showing low purityafter cation exchange. Arrow indicates mycodulcein band.

HIC Assessment.

Cell lysate was also subjected to hydrophobic interaction chromatography(HIC) using HiScreen CaptoButyl column (Cytiva, Sweden); equilibrationwas carried out using 5 column volumes of 50 mM sodium phosphate, 1Mammonium sulfate at pH 7.0. Cell lysate was then loaded into the columnat flow rate of 3 mL/min. Elution of bound proteins was performed by adecreasing ammonium sulfate gradient from 1 to 0 M using 50 mM sodiumphosphate at pH 7.0. All obtained fractions were analyzed by SDS-PAGE.

FIG. 7B shows two eluted fractions collected during the gradient elutionfrom the HiScreen Capto Butyl column analyzed on SDS-PAGE. Lane 1 showseluted fraction 1 not containing mycodulcein and Lane 2 shows elutedmycodulcein. The purity of the eluted fraction was determined byGelAnalyzer to be ˜86%.

SEC Assessment.

The eluted fraction containing native mycodulcein was then furtherpurified using size exclusion chromatography (SEC) HiPrep 26/60Sephacryl S-200 HR column (Cytiva, Sweden), and eluted with buffercontaining 50 mM sodium phosphate and 150 mM NaCl at pH 7.0. Fractionswere collected and were then concentrated and desalted using 3 kDamolecular weight cut-offs (MWCO) centrifugal filters (Millipore-Sigma,Germany) and then analyzed by SDS-PAGE.

Summary: although native mycodulcein binds successfully to the weakcation exchanger Capto MMC, the relatively low purity of the elutedfraction made CIEX a less favorable capture/intermediate purificationstep. On the other hand, a higher purity fraction was obtained from theHIC, Capto Butyl column. As per the SDS-PAGE analysis, impurities seemedto have a relatively high molecular weight which made SEC a greatcandidate to obtain a high pure native mycodulcein.

Purity after HIC/SEC is approximately 98% by GelAnalyzer of SDS-PAGE.FIG. 7C shows the eluted protein from the HIC column afterchromatographing on HiPrep 26/60 Sephacryl S-200. Lane 1 shows purifiedhis-tag mycodulcein and Lane 2 shows purified native mycodulcein.

The purified native protein purified from S. cerevisiae was tasted at0.03 mg/ml by a trained sensory scientist (0.2 mL aliquot) and found tohave a sweetness equivalent to 8° brix (approximately 8% sugarsolution). The sweet taste was very noticeably sweet, with a “clean”sweetness (sugar-like taste) with no other flavors, with a slightlydelayed onset and a sweet aftertaste.

Example 12 (Applications Data)

His-tagged mycodulcein prepared as in Example 5 and purified asdescribed in Example 5 was tested in a yogurt base. The yogurt base hadthe following recipe (Table 5):

TABLE 5 Ingredient % Water  81.1200% Pea Protein  10.6000% Sunflowerlecithin  0.0600% Coconut Oil (AAK)  5.1000% Ticaloid YG LP  0.2000%(TIC GUMS) Citri-Fi Citrus Fiber  0.6000% Canola Oil  1.0000% Cane Sugar 1.3000% YoFLEX YF-LO2  0.0200% Cultures (CHR-Hansen)  0.0000% Total100.0000%

The cane sugar is added as a carbon source for the yogurt cultures andis at least partially consumed by the cultures. Mycodulcein was added toapproximate the sweetness from 8° to 10° Brix of sugar, finalconcentration in the yogurt base is 0.05 mg/ml. Taste testing wasperformed by a trained sensory scientist and the yogurt was found tohave a sweetness equivalent to 8° brix (approximately 8% sugarsolution). The sweet taste was very noticeably sweet, with a “clean”sweetness (sugar-like taste) with no other flavors, with a slightlydelayed onset and a sweet aftertaste.

His-tagged mycodulcein prepared as in Example 5 and purified asdescribed in Example 5 was tested in whole milk; non-dairy pea-basedmilk (water, 93.75%, pea protein, 4.2%, canola oil, 1.7%, TIC Gum BlendPro 181 AG (Acacia+Gellan) 0.3%, sunflower lecithin 0.05%); cold coffee;and water (control) at a final concentration of 0.04 mg/ml, predicted toprovide a sweet level of between 8° to 10° Brix of sugar. It wasconfirmed by taste testing that the sweet protein delivers a sweet levelof between 8° to 10° Brix in all samples, and all samples had sweetnessintensity, onset, and duration that was similar to the water control.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and “at least one” andsimilar referents in the context of describing the invention (especiallyin the context of the following claims) are to be construed to coverboth the singular and the plural, unless otherwise indicated herein orclearly contradicted by context. The use of the term “at least one”followed by a list of one or more items (for example, “at least one of Aand B”) is to be construed to mean one item selected from the listeditems (A or B) or any combination of two or more of the listed items (Aand B), unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. The term “consisting of” isconstrued as a close-ended term (i.e., excluding components or stepsother than those listed). The term “consisting essentially of” allowsfor the inclusion of components or steps that are not essential to thefunction or activity of the product or method and do not materiallyaffect the function or activity.

Recitation of ranges of values herein are merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range, unless otherwise indicated herein, and eachseparate value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”) provided herein, isintended merely to better illuminate the invention and does not pose alimitation on the scope of the invention unless otherwise claimed. Nolanguage in the specification should be construed as indicating anynon-claimed element as essential to the practice of the invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

The invention claimed is:
 1. An isolated polynucleotide encoding apolypeptide selected from the group consisting of: (a) a polypeptidecomprising the amino acid sequence of SEQ ID NO: 3; and (b) apolypeptide comprising an amino acid sequence having at least 95%sequence identity to the amino acid sequence of SEQ ID NO: 3; whereinthe polypeptide has sweet-taste modulation activity, and wherein theisolated polynucleotide is operably linked to a heterologous regulatoryelement.
 2. The isolated polynucleotide of claim 1, wherein thepolynucleotide sequence further encodes a protein tag or label.
 3. Theisolated polynucleotide of claim 2, wherein the protein tag is ahistidine tag.
 4. An expression cassette comprising the isolatedpolynucleotide of claim
 1. 5. A vector comprising the isolatedpolynucleotide of claim
 1. 6. A host cell transformed with the vector ofclaim
 5. 7. A method of producing a protein having sweet-tastemodulation activity, comprising culturing the host cell of claim 6 in amedium under conditions that result in producing the protein havingsweet-taste modulation activity.
 8. A composition, comprising acombination of: (a) a product for oral administration, wherein theproduct is not Mattirolomyces terfezioides truffle, and (b) a sweeteningcomposition comprising an isolated protein produced by the method ofclaim 7, wherein the combination has enhanced sweet taste compared tothe product for oral administration.
 9. A method for modulating thetaste of a product for oral administration, comprising: combining aproduct for oral administration with an effective amount of a sweeteningcomposition comprising an isolated protein produced by the method ofclaim 7, wherein the product for oral administration is notMattirolomyces terfezioides truffle, and wherein the combination hasenhanced sweet taste compared to the product for oral administration.10. The composition of claim 8, wherein the product for oraladministration is a food, a beverage, a dietary supplement composition,or a pharmaceutical composition.
 11. The composition of claim 8, whereinthe product for oral administration is a food product selected from thegroup consisting of baked goods; sweet bakery products, pre-made sweetbakery mixes for preparing sweet bakery products; pie fillings and othersweet fillings, gelatins and puddings; frozen desserts; yogurts; snackbars; bread products; pre-made bread mixes for preparing bread products;sauces, syrups and dressings; sweet spreads; confectionary products; andsweetened breakfast cereals.
 12. The composition of claim 8, wherein theproduct for oral administration is a beverage product selected from thegroup consisting of carbonated beverages; non-carbonated beverages; andbeverage concentrates.
 13. The method of claim 9, wherein the productfor oral administration is a food, a beverage, a dietary supplementcomposition, or a pharmaceutical composition.
 14. The method of claim 9,wherein the product for oral administration is a food product selectedfrom the group consisting of baked goods; sweet bakery products,pre-made sweet bakery mixes for preparing sweet bakery products; piefillings and other sweet fillings, gelatins and puddings; frozendesserts; yogurts; snack bars; bread products; pre-made bread mixes forpreparing bread products; sauces, syrups and dressings; sweet spreads;confectionary products; and sweetened breakfast cereals.
 15. The methodof claim 9, wherein the product for oral administration is a beverageproduct selected from the group consisting of carbonated beverages;non-carbonated beverages; and beverage concentrates.
 16. A method forpurifying a protein having sweet-taste modulation activity comprising:(a) producing the protein by the method of claim 7, (b) purifying theprotein via hydrophobic interaction chromatography (HIC) followed bysize exclusion chromatography (SEC).
 17. The isolated polynucleotide ofclaim 1 comprising the nucleic acid sequence of SEQ ID NO:
 2. 18. Theisolated polynucleotide of claim 3, wherein the polypeptide comprisesthe amino acid sequence of SEQ ID NO: 21 or SEQ ID NO:
 24. 19. Theisolated polynucleotide of claim 18 comprising the nucleic acid sequenceof SEQ ID NO: 20 or SEQ ID NO: 23.